Journal of Plankton Research Vol.17 noJ pp.1079-1094, 1995 Phytoplankton exudation: exploitation of the microbial loop as a defence against algal viruses Alexander G.Murray 1 Department of Oceanography, Texas A&M University, College Station, TX 77843, USA 'Current address: CSIRO Division of Fisheries, GPO Box 1538, Hobart, Tas 7001, Australia Abstract. Healthy phytoplankton cells exude dissolved organic matter (DOM). In a model, DOM exudation is demonstrated to be a cost-effective, indirect, means of reducing vims infection, which can be a major cause of phytoplankton mortality. Diffusion theory shows that, for a given biomass, small particles will have a much higher rate of adsorption of solutes than will large ones. Thus colloidal viruses are far more likely to come into contact with bacteria than with phytoplankton if the same biomasses are present Bacteria can destroy viruses in a large proportion of contacts. Although flagellate protozoa have lower contact rates with viruses, they may consume more of the viruses that they do encounter and they deal with larger viruses particularly effectively. The exuded DOM supports bacterial growth, which in turn may support flagellates. Even fairly low levels of exudation can maintain the biomass of bacteria or small flagellates required to remove >50% of viruses before they have a chance to infect their host, at least for larger phytoplankton. High rates of virus removal may occur at the high exudation rates that are typical of late blooms. It is concluded that healthy phytoplankton cells exude DOM in order to remain healthy. Introduction Healthy phytoplankton cells exude dissolved organic matter (DOM) (Bjomsen, 1988). Although the amount exuded is a small proportion of primary production, usually less than 5% (Williams, 1990), the dominance of phytoplankton production over most of the Earth's surface makes this exudation a significant global carbon flow with important implications for marine ecosystems (Williams, 1984, 1990). The proportion of fixed carbon exuded may be far higher particularly in oligotrophic areas (Fogg, 1983). Exudation may enable phytoplankton to dispose of excess production under varying light levels which periodically overwhelm the cells' ability to process fixed carbon (Fogg, 1983; Wood and van Valen, 1990). An actively saprophytic bacterial population may help to retain nutrients in the mixed layer of the ocean (Williams, 1984, 1990). Cyanobacteria-bacteria associations fix nitrogen in freshwater, but cyanobacterial blooms are a feature of freshwater, not marine, systems (Paerl, 1988). In brief, the phytoplankton appear to gain no advantage by exuding material (Bjornsen, 1988), rather it would seem detrimental. Most obviously, the process is simply wasteful, particularly if nitrogen-containing amino acids are lost (Fogg, 1983; Fuhrman, 1987). More seriously for the phytoplankter, bacteria can take up the exudates and then compete, at an advantage, for nutrients (Cole, 1982; Bratbak and Thingstad, 1985). Some bacteria can even lyse phytoplankton cells on contact (Cole, 1982). It seems that, in the context of the current marine ecosystem paradigm, exudation really does 'make very little sense' (Bratbak and Thingstad, 1985). However, exudation does 'make sense' if the role of viruses is considered. Although most marine viruses are small (<60 nm) bacteriophages (Cochlan et al., 1993; Bratbak C Oxfoid University Press 1079 at University of Sussex on October 14, 2012 http://plankt.oxfordjournals.org/ Downloaded from
Transcript
Journal of Plankton Research Vol.17 noJ pp.1079-1094, 1995
Phytoplankton exudation: exploitation of the microbial loop as adefence against algal viruses
Alexander G.Murray1
Department of Oceanography, Texas A&M University, College Station, TX 77843,USA
'Current address: CSIRO Division of Fisheries, GPO Box 1538, Hobart, Tas 7001,Australia
Abstract. Healthy phytoplankton cells exude dissolved organic matter (DOM). In a model, DOM exudation isdemonstrated to be a cost-effective, indirect, means of reducing vims infection, which can be a major cause ofphytoplankton mortality. Diffusion theory shows that, for a given biomass, small particles will have a muchhigher rate of adsorption of solutes than will large ones. Thus colloidal viruses are far more likely to come intocontact with bacteria than with phytoplankton if the same biomasses are present Bacteria can destroy virusesin a large proportion of contacts. Although flagellate protozoa have lower contact rates with viruses, they mayconsume more of the viruses that they do encounter and they deal with larger viruses particularly effectively.The exuded DOM supports bacterial growth, which in turn may support flagellates. Even fairly low levels ofexudation can maintain the biomass of bacteria or small flagellates required to remove >50% of viruses beforethey have a chance to infect their host, at least for larger phytoplankton. High rates of virus removal mayoccur at the high exudation rates that are typical of late blooms. It is concluded that healthy phytoplanktoncells exude DOM in order to remain healthy.
Introduction
Healthy phytoplankton cells exude dissolved organic matter (DOM) (Bjomsen, 1988).Although the amount exuded is a small proportion of primary production, usually lessthan 5% (Williams, 1990), the dominance of phytoplankton production over most ofthe Earth's surface makes this exudation a significant global carbon flow withimportant implications for marine ecosystems (Williams, 1984, 1990). The proportionof fixed carbon exuded may be far higher particularly in oligotrophic areas (Fogg,1983).
Exudation may enable phytoplankton to dispose of excess production under varyinglight levels which periodically overwhelm the cells' ability to process fixed carbon(Fogg, 1983; Wood and van Valen, 1990). An actively saprophytic bacterial populationmay help to retain nutrients in the mixed layer of the ocean (Williams, 1984, 1990).Cyanobacteria-bacteria associations fix nitrogen in freshwater, but cyanobacterialblooms are a feature of freshwater, not marine, systems (Paerl, 1988). In brief, thephytoplankton appear to gain no advantage by exuding material (Bjornsen, 1988),rather it would seem detrimental. Most obviously, the process is simply wasteful,particularly if nitrogen-containing amino acids are lost (Fogg, 1983; Fuhrman, 1987).More seriously for the phytoplankter, bacteria can take up the exudates and thencompete, at an advantage, for nutrients (Cole, 1982; Bratbak and Thingstad, 1985).Some bacteria can even lyse phytoplankton cells on contact (Cole, 1982). It seems that,in the context of the current marine ecosystem paradigm, exudation really does 'makevery little sense' (Bratbak and Thingstad, 1985).
However, exudation does 'make sense' if the role of viruses is considered. Althoughmost marine viruses are small (<60 nm) bacteriophages (Cochlan et al., 1993; Bratbak
et al., 1990) larger (>60 nm) phytoplankton viruses are present in very significantnumbers, sometimes such viruses number >107 ml"1 during phytoplankton blooms(Bratbak et al., 1990). Many species of eukaryotic algae contain viral particles (vanEtten et al., 1991). A high proportion of cyanobacteria contain viruses, which are amajor cause of their mortality (Proctor and Fuhrman, 1990). Phaeocystis, Emilianiahuxleyi and Aureococcus anophagefferens blooms decline as a result of cell lysis,which can be induced by viruses (van Boekel et al., 1992, Bratbak et al., 1993 andMilligan and Cosper, 1994, respectively). Viruses can also limit algal production whenthey are concentrated from the marine environment (Suttle et al., 1990; Suttle, 1992;Peduzzi and Weinbauer, 1993) and can limit the biomass of individual algal strains(Cottrell and Suttle, 1991). Viruses are thus an important influence on phytoplanktonecology.
I suggest that phytoplankton exudation indirectly protects phytoplankton fromviruses. The mechanism by which exudation does this is by supporting the growth ofbacteria. Many bacteria kill viruses (Fujioka et al., 1980; Toranzo et al., 1982) and as adiffusion model showed small organisms have far higher contact rates, per unit bomass,than large ones (Murray and Jackson, 1992). Heterotrophic flagellates also removeviruses, particularly the larger ones (Mtfse et al., 1970; Gonzalez and Suttle, 1993). Thediffusion model of Murray and Jackson (1992) will be extended to consider theinteractions of different organisms as virus interceptors by asking two questions. Canbacteria or flagellates (for simplicity 'flagellates' means 'heterotrophic flagellates'throughout this paper) cause significant destruction of viruses? To what extent canphytoplankton stimulate local growth of bacteria or flagellates? If DOM exudationleads to a reduction in phytoplankton losses from infection greater than the cost ofexudation, then there is a cost-effective defence (Figure 1).
Method
The model of Murray and Jackson (1992) is used to calculate the rate of contact ofviruses with cells, taking into account the swimming behaviour of those cells. Virusesare small colloidal particles which may be treated as dissolved by this model. Virusescome into contact not only with their phytoplankton hosts, but also with otherorganisms. The organisms a virus is most likely to be intercepted by, before it reaches
vim*
DOMExudation Bacteria FUfleUate
Fig. 1. Nanecology. If DOM exudation results in a reduction in phytoplankton losses due to infection greaterthan the cost of that exudation, then there is a cost-effective anti-vinis defence.
its host, are bacteria and flagellates, collectively called 'interceptor organisms'. Themodel is expanded to consider the chance of a virus coming into contact with aninterceptor organism relative to the chance of it reaching a host phytoplankton cell. Theeffect, on interception, of reducing the concentration of viruses in the vicinity of a host,due to its own removal of viruses, is explored.
The second part of the modelling is the consideration of biological factors on virusinterception. First, the mortality rate of viruses per contact with interceptors isconsidered. This rate converts interception into actual destruction of viruses. Thenequations are presented to estimate the biomasses of both bacteria and flagellates basedon phytoplankton exudation. Using contact rate, mortality per contact and relativebiomass, the effectiveness of the antiviral shield can be calculated.
Particle dynamics
The contact rate of viruses with spherical cells (Murray and Jackson, 1992) is:
c = 2ndDvSh (1)
where d is cell diameter, Dv is viral diffusivity and Sh is the Sherwood number (thesymbols used in this paper are summarized in Table I). Sh describes the effect of fluidmotion and depends on the Peclet number (Pe). Pe depends on the characteristic lengthand the characteristic velocity. For motile phytoplankton cells, characteristic length is dand velocity is swimming velocity (w). Pe and Sh can be approximated by:
Sh = 0.5[l + ( l+Pe) 1 / 3 ] (2)
and
Pe = dw/Dv. (3)
Swimming velocity, w, may be a function of d, e.g. 10 d s"1, in which case contact ratedepends only on host particle size and viral diffusivity. It follows that the relative viralcontact rates of two separate cells depend only on d and Sh (and hence w and Dv). Viraldiffusivity is temperature and viscosity dependent (Berg, 1983), but when these arefixed it can simply be related to the inverse of viral diameter. At 283 K in water, therelationship is (Murray and Jackson, 1992):
D v « 3 . 1 6 x 10"13 c m V 1 / * , (4)
The virus can come into contact with two types of cells: intercepting bacteria orflagellates, and host phytoplankton. The rate of viral contact with an interceptingbacterium or flagellate, collectively i, relative to the contact rate for the virus with itsphytoplankton host, h, is called a. It can be derived from Eq.(l)
•DimensionJess, but units used in derivation shown to aid comprehension.
The relative rate of contact with viruses for equal biovolumes of spherical interceptororganisms and host phytoplankton is called p. This can be calculated by dividing a byd? and multiplying by d^, where dt and dh are interceptor and host diameters,respectively.
p = (6)
One problem with bacterial interception of viruses is that the virus concentration in thevicinity of a host cell is reduced due to adsorption by that phytoplankton cell. Thisreduces contact of viruses with bacteria or flagellates in the immediate vicinity of thevirus' host If a host is a perfect adsorber and fluid is motionless, then die localconcentration of viruses is (Berg, 1983):
-0.5d/r) (7)
where r is the distance from the cell centre and vx is the concentration of viruses at agreat distance from the host Owing to fluid shear rate, E, the radius of virus depletion
is limited to a radius r , ^ (Bowen et al., 1993). Beyond this radius, background virusconcentrations exist, so if r > = Tmm then v = v^.
rmax = (Dv/E)°'5 (8)
Biological modelling
Interference in virus transmission depends not only on the physics of the relativecontact rate, but also on biology. The product of mortality rate per contact (m) andrelative contact rate (Eq. 6) gives the number of viruses removed by interceptororganisms before one virus comes into contact with a phytoplankter if interceptor andphytoplankton have equal biovolumes:
/ = mp (9)
The protection actually accorded by bacteria, or flagellate, depends on the localbiomass of these organisms relative to the phytoplankton biomass. Ideally, the ratio ofthe biovolume, not biomass, should be used; however, the variation in the densities ofcells makes calculation of this uncertain.
Jb = IB/P (10)
Jf = IF/P (11)
Bacterial biomass can be raised above background levels if phytoplankton provide asubstrate for growth. They do this by exuding labile DOM as a proportion of primaryproduction, x, multiplied by biomass P. Bacteria must be able to take up this DOM and,with reasonable efficiency, b, convert it to bacterial biomass if viruses are to beintercepted. Exudation and the production efficiency of bacteria determine relativeproduction of bacteria to phytoplankton. Finally time scales, i.e. the turnover rate ofbacteria, tb, relative to phytoplankton, tp, determine biomass:
B = bxPtb/tp (12)
Flagellate biomass depends on bacterial production (bxP), efficiency of flagellategrowth (f) and turnover time relative to phytoplankton turnover time (tf/tp):
F =JbxPtf/tp (13)
Phytoplankton turnover time can be related to body carbon or volume (Banse, 1976)and hence to cell diameter
Consider the characteristics of the organisms that control the physical model. Viraldiffusivity, £>„, is a constant for a given virus at a constant temperature. Viruses thataffect algae tend to be large and thus less diffusive than bacteriophages. Van Etten et al.(1991) gave a range from 22 to 400 nm, giving diffusion coefficients of1.6 x 1 0 ~ 7 - 8 x 10~9 cm2 s~' (Eq. 4). From this, a 200 nm virus withDv = 1.6 x 10~8 cm2 s"1 would be typical. The diameter of viruses, and thusdiffusivity, is related to the diameter of the host (Murray and Jackson, 1992). Thediameter, d, of phytoplankton ranges from <0.5 p,m to >1 cm (ElbrSchter, 1984).Flagellates range from <1 to 100 u,m in diameter (Fenchel, 1987). Very smallflagellates (<1 JJLITI) may be important grazers on bacteria (Fuhrman and McManus,1984) and hence may be indirectly supported by exudation. Marine bacteria are small,generally <1 u,m long; however, bacteria that feed on phytoplankton exudates tend tobe larger than most, d ss 0.6 (Painting et al., 1989). Williams (1984) noted that thediameter of bacteria could be <0.2 u,m, but suggested a mean value of 0.5 u,m foroffshore bacteria and 0.6 u-m for coastal forms.
The model of Murray and Jackson (1992), which is used to calculate virus:particlecontact rates, was derived to consider the contact of phytoplankton or bacteria withviruses. Possibly active grazers such as flagellates could increase contact above thisbasic rate by detecting and then actively intercepting viruses. Formulae for the contactrate between virus-sized particles and flagellates have been derived by Shimeta(1993). The contact rates of typical algal viruses of 20-400 nm diameter and a 4 u,mflagellate swimming at 200 \ixt\ s~' were calculated from the sum of Shimeta's equations(2) and (3) and by Eq. 1 (this paper). For virus-sized particles, the equations produce nearlyidentical results (Figure 2).
The contact rate per individual increases faster than the host's radius, and per unitbiomass contact decreases faster than the host's radius (Figure 3). As a result, for twoorganisms whose diameters differ by an order of magnitude, the smaller has a verysmall relative contact rate per individual, while the larger has a very small relative
1260
Fig. 1 Contact rale between nanometer-sized particles and a 4 \ua flnyllate swimming at 200 |im s '. Thesecontact rates are calculated using the models of Murray and Jackson (1992) and Shimeta (1993). The modelsagree well for the range 20-400 nm typical of algal viruses.
Fig. 3. Contact rate per cell and per unit biomass for plankton cells with viruses of diffusivity 1.25 x KT8.Contact rate per cell, a, is relative to a 100 (jun plankter, per unit biomass, f}, is relative to a 0.1 jun plankter.
contact rate per unit biomass. It is on the basis of the latter observation that the use ofbacteria as an anti-viral shield becomes possible. Swimming speed, in body lengths persecond, tends to decrease with body length. This is particularly true for flagellates,which may swim at 200 urn s"1 regardless of d (Fenchel, 1987). A simple directrelationship between swimming speed and body length therefore minimizes contactrates for smaller organisms, (conservatively) underestimating their effectiveness asvirucides. For more details on the contact model, see Murray and Jackson (1992).
Host, interceptor and virus spatial interactions
For bacteria to intercept viruses, they must be able to maintain themselves in thevicinity of the host, or at least of a patch of closely related phytoplankton cells.However, their efficiency as a virucides is reduced if they are very close to the host cellbecause some viruses would be adsorbed by that cell. Therefore, effective defencerequires that not too large a proportion of bacteria is very close to the cell. Sinceflagellates arc more powerful swimmers than bacteria, their distribution will be able toshadow that of the bacteria.
There is evidence that bacteria can cluster in the vicinity of phytoplankton cells in theregion of enhanced exudate concentration known as the phycosphere (Azam andAmmerman, 1984). Bacteria may be able to concentrate in the phycosphere in spite ofshear (Bowen et al., 1993) and slow swimming of hosts of >2 jtm diameter (Jackson,1987). However, even when bacteria cluster around cells >80% of the population areoutside the phycosphere (Bowen et al., 1993). Therefore, bacteria probably only clusteraround large individual cells or colonial forms. Mucus of Phaeocystis colonies cansupport bacterial concentrations 2-11 times that of water on the same slide (Putt et al.,1994). Diatoms can also support bacteria in a slime layer surrounding the cell (Bratbaket al., 1990).
Individual smaller cells might not be able to exude sufficient large DOM to allowbacteria to accumulate in their vicinity (Jackson, 1987). In a patch of cells, however,this is not a problem. Bacterial biomass correlates well with chlorophyll concentration
(Fuhrman et al., 1980). Phytoplankton blooms are closely associated with bacteria(Paerl, 1988), which form distinctive communities (Martin, 1980; Painting etal., 1989).A patch is likely to be particularly vulnerable to viral infection because of the closenessof many genetically similar cells (Cottrell and Suttle, 1991). Any anti-viral protection,even at the relatively low level possible for smaller phytoplankton, would be beneficialwhen patch members are relatives. As will be discussed later, exudation rates are higherin blooms (Martin, 1980). Owing to the tight coupling of DOM production byphytoplankton and uptake by bacteria (Fuhrman, 1987), any patch of phytoplanktonthat persists should be able to support bacterial growth.
Viruses are less concentrated very close to the host cell due to their adsorption by thehost The bacteria very close to a phytoplankton cell would therefore adsorb fewerviruses (Figure 4). This zone will, for convenience, be referred to as the 'avirosphere'.Adsorption by phytoplankton means that viral concentration is 50% of backgroundlevels atr = d and 75% at r = 2d. These radii enclose 8 or 64 cell volumes. Thus, for atypical phytoplankton, biovolume concentration of 1 p.p.m. (Elbrachter, 1984) theavirosphere represents <0.01% of the fluid volume. It would be difficult for most of thefree-swimming bacteria to concentrate into such a small volume, but at higherphytoplankton concentrations the volume contained within the combined avirospheresmight be large enough to significandy affect interception of viruses by bacteria andflagellates.
With shear the radius of the avirosphere becomes restricted, ranging between 1 and10 u.m for Dv = 1.25 x 10~8 and typical oceanic shear rates (0.5-0.005 s"1) (Bowen etal., 1993). Since solutes are about two orders of magnitude more diffusive than viruses(Murray and Jackson, 1992), the radius of the avirosphere is 0.1 (and the volume 0.001)that of the phycosphere in the presence of shear, r ^ , (Eq. 8). So small a volume isunlikely to have much effect on viral interception by bacteria
Swimming has even stronger effects on the transmission of solutes (Jackson, 1987)and viruses (Murray and Jackson, 1992), and would further reduce the avirosphere.Phytoplankton can also chemically discourage colonization of their surface (Kellam
Fig. 4. The effect of ho5t absorption of viruses on local viral concentration in an undisturbed medium. Thezone of >25% viral depletion is referred to as the 'avirosphere'.
and Walker, 1989). Clustering of bacteria within the avirosphere of motilephytoplankton is therefore very unlikely to be enough to seriously reduce the bacteria'seffectiveness as virucides.
Effect of contact on viruses
In this section, the mortality per contact parameter m is evaluated. The mortality rate ofviruses per contact with either bacteria or flagellates, is of the order 0.1—1. Rates maybe less for native marine viruses in contact with bacteria or for small viruses being eatenby flagellates.
Bacteriophages are a major source of bacterial mortality (Proctor and Fuhrman,1990), so it would be in the interest of bacteria to have anti-viral defences. Bacteria canbreak down the constituent proteins (Hollibaugh and Azam, 1983) and nucleic acids(Ammerman and Azam, 1991) of viruses. Observed removal rates of virus by bacteriawere 19-250 |xm3 s"1 (Toranzo et al, 1982; Heldal and Bratback, 1991), whichcompares well with Murray and Jackson (1992) model estimates of 18-100 jim3 s"1.Observed viral decay rates (Carlucci and Pramer, 1960; Metcalfe et al., 1974; Zachary,1976; Fujioka et al., 1980) were used with the same model to estimate the size ofpopulations of 0.6 \LTCI diameter bacteria: 0.06-7 x 105 cm"3. Typical observedpopulations are 0.2-20 x 105 cm"3 (Williams, 1984). This would suggest m « 0.3;however, estimation of m is very sensitive to bacteria diameter. If bacteria are 1 \i.m,then the estimate of m = 0.15. As 1 u.m is very large for marine bacteria, m = 0.15 maybe taken as a minimum of virus destruction per contact. Viruses may be deactivated byenzymes released from bacteria, even though the viruses are not in the immediatevicinity of the source (Toranzo et al., 1982). However, fine filtration or antibiotictreatment removes much of the virucidal activity of sea water (Fujioka et al., 1980;Girones et al., 1989) and therefore the presence of bacteria is probably required for viralremoval. As model virus:bacteria contact rate estimates are in good agreement withobservations, it is likely that m is fairly high. There is a caveat: most of the works citedinvolved human enteric viruses or coliphages. Only one referred to native martinephages (Zachary, 1976) and for this viral population the decay rate appears to be lowerthan those of most exotic (to the marine environment) phages. Suttle and Chen (1992)also found native marine viruses to be more weakly affected by bacteria than othercauses of mortality.
Although larger organisms have lower contact rates per unit biomass, grazers may bemore adept at dealing with viral particles that they intercept. Protozoa can graze virusesin culture, greatly increasing their mortality (MSse et al., 1970; Gonzalez and Suttle,1993). In natural seawater, flagellates appeared to have more impact on marine virusesthan bacteria had (Suttle and Chen, 1992). Flagellates are equipped to remove smallparticles from the water (Marchant and Scott, 1993), whereas bacteria may simply repelat least some viruses. Although choanoflagellates can retain particles of 0.063 jimdiameter, the uptake rate is considerably smaller for 0.25 Jim (virus-sized) than for0.5 Jim (bacteria-sized) particles (Marchant and Scott, 1993): 1.3 versus 3 nl cell"1 h"1.Choanoflagellates are fairly effective grazers on virus-sized particles and may evenconcentrate exclusively on them at times (Gonzalez and Suttle, 1993). Suttle and Chen(1992) found other heterotrophic flagellates to be fairly inefficient grazers of virus-
sized particles. Many flagellates ingest small viruses at about -4% of the rate at whichthey ingest bacteria, but deal more effectively with larger viruses (Gonzalez and Suttle,1993). Larger viruses are typical of phytoplankton (Bratbak et al., 1993) and largerviruses are associated with larger species (Murray and Jackson, 1992). The parameter mis often fairly high since retention of viruses is significant at least for some flagellatespecies and for larger viruses. Indeed, the largest viruses are known only from thefeeding vacuoles of protozoa (Gowing, 1993).
Biomass of bacteria and flagellates supported
The biomass of bacteria and flagellates that could be supported by exudation have beenestimated using model equations. Roughly, production by uptake of labile exudates is2-15% (bacteria) and 0.7-7% (flagellates) of phytoplankton production. Given similarturnover times of bacteria, flagellates and phytoplankton, these levels of productionrepresent relative biomasses. Fogg (1983) estimated that exudation could support aheterotroph biomass of up to 25% of phytoplankton biomass.
The biomass of bacteria relative to phytoplankton depends on their relativeproduction and turn over times. Williams (1984) estimated marine bacterial generationtimes at 12-24 h, although possibly only a small fraction, rapidly turning over, isactually active. In the <3 \i,m phytoplankton fraction in Baltic coastal waters, turnoverranged from 4.5 to 33 h (Larsson and HagstOrm, 1982). Total bacterial turnover is thussimilar to that of smaller phytoplankton, but may be faster than that of largerphytoplankton. The exponent of the relationship of turnover time to cell weight is 0.04-0.25 and to volume slightly higher—0-0.2 (Banse, 1976)—giving a relationship to d°-d06 or n = 0-0.6.
The turnover rate depends on the environment Two of the four data sets analysed byBanse (1976) had exponents close to zero and were therefore size-independentDinoflagellates show little evidence of any dependence of growth rate on size(ElbrSchter, 1984). If turnover of bacteria is faster than that of phytoplankton, thenbiomass will be lower per unit production and vice versa. If turnover times are similar,B is simply the product of the growth efficiency of bacteria and the labile component ofphytoplankton exudation, i.e. the time element is removed.
Exudation rates vary with both phytoplankton type and environment (Lancelot andBillen, 1985). Fogg (1983) suggests exudation ranges from 5% of primary productionin eutrophic waters to 40% in oligotrophic ones. Williams (1990) concluded thatexudation was usually <5% of primary production for exponentially growing cells, buthigher when nutrients were limiting. Coupling of bacterial uptake and phytoplanktonexudation of simple compounds is high (Cole, 1982; Fuhrman, 1987; Painting et al.,1989). Small molecules amount to half or more of exudate (Lancelot, 1984; Lignell,1990), and since they can be taken up directly by bacteria this may be considered thelabile fraction. The late period of a bloom, before cell death occurs, may see a period ofhigh exudation rates (Martin, 1980; Sell and Overbeck, 1992). Similarly, the late part ofa bloom following an upwelling is a time of very high bacterial biomass (Painting et al.,1993). In the late part of a bloom, diatoms form an extracellular 'slime subsystem'which supports bacteria close to the algal cell, but not on its surface (Bratbak et al.,1990). High bacteriophage populations in the 'slime subsystem' suggest that anti-viral
activity would be strongly selected for in associated bacteria. High exudation whennutrients are limited would be advantageous if its purpose were to provide viralprotection: since the cells cannot grow, production should be switched to protection.
Smaller algae are theoretically more vulnerable to viral infection. Smaller organismshave a higher probability of containing viruses (Proctor and Fuhrman, 1990). Tocounteract this threat, it is logical that smaller phytoplankton should have higherexudation rates. A re-analysis of data presented by Larsson and Hagst5rm (1982)showed a very close relationship between size fractions of primary production and theproportion exuded (Figure 5). If this regression of proportion of production by >3 p-malgae versus exudation is followed, then if 100% primary production is by >3 p,morganisms 2.6% is exuded; if 0%, 31 % is exuded. There is no co-variation betweenproduction and phytoplankton size, so this relationship between exudation and size isnot a coincidental variation of both exudation and phytoplankton size with nutrientstress. Exudation is indeed higher for smaller phytoplankton.
Bacterial growth efficiencies on glucose and amino acids are typically 50-90%(Williams, 1984). Therefore bacterial production, b, is probably at least half ofphytoplankton exudation of labile organic matter (Lignell, 1990). If the labileproportion of exudate is -0.66, then exudation of 5-̂ 40% of production means organicmatter available for bacterial uptake, x = 0.03-0.3. If b ss 0.66, then 5% exudationshould support a bacterial biomass of about 2% of die phytoplankton biomass, and 40%exudation about 15% of phytoplankton biomass.
Flagellate numbers can increase rapidly in the late bloom period (Painting et al.,1989; Marchant and Scott, 1993). Biomass is often highest at the time of greatestphytoplankton production, i.e. before senescence (Paerl, 1988). Flagellate growth israpid, enabling them to keep pace with bacterial production (Fenchel, 1984, 1987).Therefore flagellate biomass per unit production should be similar to that of smallphytoplankton, but perhaps less than that of large phytoplankton (Banse, 1976). Othergrazers have difficulty in utilizing bacteria (Fenchel, 1984; Shimeta, 1993) so small,
0 10 20 30 40 SO 60 70 80 90 100
% Production > 3 um
Fig. 5. Exudation by phytoplankton relative to the proportion of primary production by >3 \im particles.Tbere a a strong negative relationship (-0.284) between exudation and proportion of production by largercells (P - 0.003). At 0% production by >3 jun cells, exudation is 31%, and at 100* production by thisfraction of the cells exudation is 2.6%.
possibly very small (Fuhrman and McManus, 1984), protozoa use most bacterialproduction. Because they are faster swimmers than bacteria, flagellates can maintaintheir position relative to any significant patch of bacteria. Experimentally, someprotozoa respond very rapidly to the presence of phytoplankton (Turley et al., 1986).Flagellate production (/) is about 40% efficient (Lucas et al., 1987). Given the bacterialproduction discussed earlier (2-15% of primary production), a flagellate biomassequivalent to 0.7-7% of phytoplankton biomass could be supported by exudation.
Combining physical and biological factors
To compare the efficiencies of viral interception, I will consider the effects of 0.25, 1and 4 u,m interceptor organisms on viral contact with motile phytoplankton. Marinebacteria are generally larger than 0.25 |xm (Williams, 1984), so this represents aminimum. Bactiverous flagellates tend to be small (Fenchel, 1984), possibly very small(Fuhrman and MacManus, 1984), so although larger bacterivores exist, 4 u,mrepresents the larger range of these grazers on bacteria. The 1 u-m size is intermediatebetween small flagellates and large bacteria.
Biomass production can only amount to a few per cent of phytoplankton biomass,and not all non-host:virus contacts are lethal. The controlling factor in antiviralprotection is the product of vinicidal effectiveness and biomass of bacteria or flagellatesdivided by phytoplankton biomass, mB/P or mF/P, which will be referred to as'effective relative biomass' (ERB). Given that B and F are in the range of 1-10% ofphytoplankton biomass, and m 0.1-1, ERB ranges from 0.001-0.1.
We can use ERB to calculate the protective effects of bacteria (Eq. 10) or flagellates(Eq. 11). At y = 1, the virus:host contact rate equals the rate of removal by interceptororganisms. Therefore, 50% of viruses are removed before they even have a chance toinfect the host. Although removal of <50% of viruses may be important, removal of>50% is undoubtedly significant if viral mortality is at all important for phytoplankton.Therefore, J = 1 can be used as a threshold of significant protection.
Knowing the relative virus interception rates of intercepting organisms and of hosts,we can calculate the value of ERB required to produce 7 = 1 , i.e. destroy half theviruses before they contact the host (Table Da). Since the maximum heterotrophbiomass supported by exudation is about 25% of autotroph biomass (Fogg, 1983) andm cannot be greater than 1, only required values of ERB of <0.25 are considered. Theminimum size of heterotroph that can receive 50% protection (i.e. maximum ERB =0.25) is 0.54, 2.46 or 10.6 u,m for intercepting organisms of, respectively, 0.25, 1 or4 fxm. At the extreme low estimate of ERB = 0.001, only smaller bacteria can providesignificant defence for most hosts. If ERB =0.01, then bacteria or small flagellates canprotect most phytoplankton. This ERB value would only require low levels ofexudation, <5%, if the bacteria or flagellates were reasonably efficient virucides. IfERB = 0.1, as might be the case in a bloom with high exudation (Martin, 1980), theneven large bacteria or small flagellates could provide protection to all but the smallestphytoplankton. Flagellates, particularly very small flagellate (Fuhrman and McManus,1984), may be a significant back-up defence if bacteria are ineffective virucides, orviruses are larger (Cochlan et al., 1993) which is typical of the viruses of largerphytoplankton (Murray and Jackson, 1992). Certainly, flagellates of >4 u,m would
Table EL Effective relative biomass (ERB) of 0.25, 1 and 4 pjn bacteria or flagellates required to remove50% of viruses before they come into contact with 1-1000 pjn motile or non-motile phytoplankton cells.ERB is the product of antiviral efficiency and biomass of interceptors, divided by the biomass ofphytoplankton: mB/P or mF/P. Values of ERB > 0.25 are considered to mean strong antiviral protection isimpossible. D = 1.25 x 10~*
p
u j n
(a) Motile phytoplankton15
1050
100500
1000
(b) Non-motile phytoplankton1
10100
1000
Bat F
0.25 |Am
0.090.0080.0030.000330.000131.4 x lCT3
5.9 x 1CT6
0.0580.000585.8 x 10"*5.8 x 10-8
1 |im
_0.0890.0330.00370.00140.000176.5 x 1(T3
-0.00656.5 x lCT3
6.5 x 10"7
4 ujn
-
__
0.030.0120.00130.00053
-0.0530.000535.3 x W6
appear to be unable to contribute much protection. Bacterial biomass may be muchgreater than that needed to establish J = 1. For a host that needs ERB of 0.01 for J = 1,an ERB value of 0.1 gives 7 = 1 0 and 10 viruses are intercepted for each one that getsthrough (90.9% protection). The protective effects of flagellates and bacteria areadditive.
Turnover time for the host phytoplankton may decrease with size, decreasing B/P forsmall phytoplankton. Turnover will decline 0.5 for each order of magnitude increase ind if n = 0.3 and 0.75 if n - 0.6 (Eq. 14). Thus, the same effective protection can beobtained for hosts of two or four times the exudation rate for each order of magnitude d> 1 \im for these values of n. As f$ increases by more than an order of magnitude foreach order of magnitude increase in d, the protective effect increases by >2.5 per unitbiomass for each 10-fold increase in diameter, even at the highest value of n. Two ofthe four sources used by Banse (1976) showed n as close to zero and thereforeprotection increases in direct proportion to diameter. The growth rate of dinoflagellatesalso does not depend on size (ElbrSchter, 1984). Since k occurs in the growth rate of allphytoplankton, bacteria and flagellates, it is cancelled out when relative growth ratesare calculated. Slower turnover of large phytoplankton may weaken, but does notfundamentally prevent, bacterial antiviral activity. Strong size dependence in thephytoplankton growth rate could rule out larger nanoflagellates as a cost-effectiveantivirus protection.
For non-motile larger phytoplankton, antiviral defence is even stronger (Table lib).Phytoplankton of 10 u,m need only a fifth of the exudation rate and 1000 \im ones only1 % of the exudation rate required to protect motile forms (Table Ha). Exudation ratesfor (non-motile) diatoms are much lower than those of motile forms (Lancelot andBillen, 1985). The avirosphere would be relatively large, slightly reducing the
effectiveness of interception. Conversely, protection would be enhanced by theincreased ability of bacteria to locate non-motile phytoplankton.
Conclusion
Phytoplankton exude DOM (Williams, 1990), the uptake of which by bacteria iscoupled closely to its production (Fuhrman, 1987). The result is bacteria can grow inphytoplankton patches, despite competing for nutrient competition (Cole, 1982;Bratbak and Thingstad, 1985) and possibly causing lysis (Cole, 1982) of thephytoplankton. I propose that a reason for phytoplankton exudation is to encouragebacteria, or flagellates, as an antiviral shield. For this to be true, three conditions mustbe met: phytoplankton viruses must be an important cause of mortality; bacteria and/orflagellates must exhibit significant virucidal properties; and phytoplankton must be ableto encourage bacteria and/or flagellate growth in their vicinity (but not at the cellsurface). All three conditions appear to hold, at least for bacteria. I therefore concludethat bacteria are a potent protection against viruses. Flagellates may provide protectionagainst the large viruses that are typical of larger algal cells (van Etten et al., 1991).
Observed exudation is not always consistent with the theory of Wood and van Valen(1990) which depends on cells switching rapidly from light to nutrient limited. Thiswould not be the case for the high exudation rates observed late in blooms, wherenutrient limitation develops gradually (Martin, 1980; Sell and Overbeck, 1992).Exudation of amino acids, even as a minority constituent, also cannot be explained bythis theory (Fuhrman, 1987). Nor is the theory of Williams (1990) that exudation is tosupport recycling always valid, since there are distinct bacterial communities associatedwith exudation and with recycling (Martin, 1980). Both these theories may be valid inmany cases, but they cannot explain all exudation (nor do they claim to). Even ifantiviral protection is not the principal reason for exudation, the effects shown in thispaper would still be a result of exudation.
Virucidal organisms are not the sole cause of the mortality of viruses; UV radiation(Suttle and Chen, 1992; Murray and Jackson, 1993) or sinking out with sediment inturbid water (Metcalfe et al., 1974) can also remove viruses. Refractory DOM mightalso be used by virucidal bacteria eventually. These processes, however, are not relatedto local phytoplankton biomass, and so as the latter rises their protective effects becomeless important and they cannot control virus epidemics in blooming populations.
Acknowledgements
This work was supported by die following grants: Office of Naval Research ContractN00014 87-K0O5 and US Department of Energy DE-FGO5-85-ER6O341. DrG.AJackson (TAMU) made this work possible. Dr S.Jeffrey (CSIRO) suggestedFigure 1 and Dr V.Mawson provided editorial assistance.
References
AmmermanJ.W. and AzanvF. (1991) Bacterial 5'-nudeotJdase activity in estuarine and coastal marinewaters: Characterization of enzyme activity. LimnoL Oceanogr., 36, 1427-1436.
AzanvF. and AmmermanJ.W. (1984) The cycling of organic matter by bacterioplankton in pelagic marineecosystems: Microenvironmental considerations. In Fashan^MJ.R. (ed.). Flows of Energy and Material inMarine Ecosystems: Theory and Practice. Plenum Press, New York, pp. 345—360.
BanseJC (1976) Rates of growth, respiration and photosynthesis of unicellular algae as related to cell size—areview. J. Pkycol., 12, 135-140.
Berg,H.C. (1983) Random Walks in Biology. Princeton University Press, Princeton, NJ.BjomsenJ'.F. (1988) Phytoplankton exudation of organic matter why do healthy cells do it? LinvwL
Oceanogr., 33, 151-154.BowenJ.D., Stolzenbach,KJ). and Chisholm,S.W. (1993) Simulating bacterial clustering around phyto-
plankton cells in a turbulent ocean. LimnoL Oceanogr., 38, 36-51.Bratbak,G. and Thingstad,T.F. (1985) Phytoplantton-bacteria interactions: an apparent paradox? Analysis of
a model system with both competition and commensalism. Mar. EcoL Prog. Ser., 25, 23-30.Bratbak,G., HeldaUd., Noriand,S. and Thingstad,T.F. (1990) Viruses as partners in spring bloom microbial
trophodynamics. AppL Environ. Microbiol., 56, 1400-1405.Bratbak,G., EggeJ.K. and HeldalJvL (1993) Viral mortality of the marine alga Emiliania huxteyi
(Haptophyceae) and termination of algal blooms. Mar. EcoL Prog. Ser., 93, 39—48.CarluccuA-F. and PramerJD. (1960) An evaluation of the factor effecting the survival of Escherichia coli in
sea water. IV Bacteriophages. AppL Microbiol., 8, 254-256.ColeJJ. (1982) Interactions between bacteria and algae in aquatic ecosystems. Annu. Rev. EcoL Syst., 13,
291-314.Cochlan,W.P., WiknerJ., Steward,G.F., Smith,D.C. and Azamjv (1993) Spatial distribution of viruses,
bacteria and chlorophyll a in neritic, oceanic and estuarine environments. Mar. EcoL Prog. Ser., 92,77-87.
Cottrell,M.T. and Suttle.C.A. (1991) Wide-spread occurrence and clonal variation in viruses which cause lysisin a cosmopolitan, eukaryotic marine phytoplankter, Micromonas pusilla. Mar. EcoL Prog. Ser., 78, 1-9.
Elbrachter,M. (1984) Functional types of marine planktonic primary producers and their relative significancein the food web. In Fasham>< J.R. (ed.), Flows of Energy and Material in Marine Ecosystems: Theory andPractice. Plenum Press, New York, pp. 191-221.
Fenchel.T. (1984) Suspended marine bacteria as a food source. In FashanvM J.R. (ed.), Flows of Energy andMaterial in Marine Ecosystems: Theory and Practice. Plenum Press, New York, pp. 301-315.
Fenchel,T. (1987) Ecology of Protozoa. Springer-Vetiag, Berlin.Fogg.G.E. (1983) The ecological significance of extracellular products of phytoplankton photosynthesis. Bot.
Mar., 26, 3-14.FuhrmanJ.A. (1987) Close coupling between release and uptake of dissolved free amino acids in seawater
studied by an isotope dilution approach. Mar. EcoL Prog. Ser., 37, 45—52.FuhrmanJ.A. and McManus.G.B. (1984) Do bacteria sized marine eukaryotes consume significant bacteria]
production? Science, 224, 1257-1259.FuhrmanJ.A., AmmermanJ.W. and Azam,F. (1980) Bacterioplankton in the coastal euphotic zone:
distribution, activity and possible relationships with phytoplankton. Mar. BioL, 60, 201-207.FujiokaJt.S., LohJ".C. and Lau,S.L. (1980) Survival of human enteroviruses in the Hawaiian ocean
environment; evidences for virus inactivating microorganisms. AppL Environ. MicrobioL, 39, 1105—1110.Girones,R., JofreJ. and Bosch^A. (1989) Natural inactjvation of enteric viruses in seawater. J. Environ. Qual,
18, 34-39.GonzalezJ.M. and Suttle.C.A. (1993) Grazing by marine nanoflagellates on viruses and viral-sized particles:
ingestion and digestion. Mar. EcoL Prog. Ser., 94, 1-10.Gowing>I.M. (1993) Large virus-like particles from vacuoles of phaeodarian radiolarians and from other
marine samples. Mar. EcoL Prog. Ser., 101, 33-43.Heldal.M. and Bratbak.G. (1991) Production and decay of viruses in aquatic environments. Mar. EcoL Prog.
Ser., 72, 205-212.HollibaughJ.T. and Azam,F. (1983) Microbial degradation of dissolved proteins in seawater. LimnoL
1253-1266.Kellam.S. and WalkerJ.M. (1989) Antibacterial activity from marine microalgae in laboratory culture. Br.
PhycoL, J. 24, 191-194.Lancelot,C. (1984) Extracellular release of small and large molecules by phytoplankton in the Southern Bight
of the North Sea. Estuarine Coastal Shelf Set, 18, 65-77.Lancelot,C. and Billen.G. (1985) Carbon-nitrogen relationships in nutrient metabolism of coastal marine
ecosystems. Adv. Aquatic MicrobioL, 3, 263-321.Larsson.U. and HagstomvA. (1982) Fractionated primary production, exudate release and bacterial
production in a Baltic eutrophication gradient. Mar. BioL, Si, 55-70.LignelUl. (1990) Excretion of organic carbon by phytoplankton: its relation to algal biomass, primary
productivity and bacterial secondary production in the Baltic Sea. Mar. EcoL Prog. Ser., 68, 85-99.Lucas,M.I., Probyn,T.A. and Painting j ' .A. (1987) An experimental study of nticroflageflate bactivory: further
evidence for the importance and complexity of microplanktonic interactions. S. Afr. J. Mar. ScL, 5, 791—808.
MarchantJIJ. and ScottJU. (1993) Uptake of sub-micrometre particles and dissolved organic material byAntarctic choanoflagcllates. Mar. EcoL Prog. Ser., 92, 59—64.
Martin.Y.P. (1980) Succession ecologique de communautes bacteriennes au cours de revolution d'unecosysteme phytoplanctonjque marin experimental. OceanoL Acta, 3, 293-300.
Metcalfe.T.G, Wallis.C. and MelnkkJL- (1974) Virus enumeration and public health assessments inpolluted surface water contributions to transmission of virus in nature. In Malina I F Jr and Sagik,B.P.(eds). Virus Survival in Water and Wastewater Systems. University of Texas Press, Austin, pp. 57-79.
Milligan,K.L.D. and Cosper,E.M. (1994) Isolation of a virus capable of lysing the brown tide microalga,Aureoccocus anophagefferens. Science, 266, 805-807.
MOseJ.R., Dostal.V. and Wege.H. (1970) Inaktivierung von Viren dutch Abwasserprotozoen. Arch. Hyg.,154, 319-330.
Murray ,A.G. and Jackson,G.A. (1992) Viral dynamics: a model of die effects of size, shape, motion andabundance of single<elled planlctonic organisms and other particles. Mar. EcoL Prog. Ser., 89, 103-116.
Murray ,A.G. and Jackson,G.A. (1993) Viral dynamics II: a model of the interaction of ultraviolet light andmixing processes on virus survival in seawater. Mar. EcoL Prog. Ser., 102, 105-114.
Painting,SJ., Lucas,M.I. and Muir.D.G. (1989) Fluctuations in heterotrophic bacterial community structure,activity and production in response to development and decay of phytoplankton in a microcosm. Mar. EcoLProg. Ser., 53, 129-141.
Paintmg.SJ., Lucas.M.I., Peterson,W T., Brown.P.C, HutchingsX. and MitcheU-Innes.B.A. (1993)Dynamics of bacterioplankton, phytoplankton and mesozooplankton communities during die developmentof an upwelling plume in the southern Benguela. Mar. EcoL Prog. Ser., 100, 35-53.
PaerlJl.W. (1988) Nuisance phytoplankton blooms in coastal, estuarine, and inland waters. LimnoLOceanogr., 33, 823-847.
Peduzzi.P. and Weinbauer^M.G. (1993) The submicron size fraction of seawater containing high numbers ofvirus particles as bioactive agent in unicellular plankton community successions. J. Plankton Res., 15,1375-1386.
Proctor ,R.M. and FuhnnanJ. A. (1990) Viral mortality of marine bacteria and cyanobacteria. Nature, 343, 60-62.
Puttjd., Miceli.G. and Stoecker,D.K. (1994) Association of bacteria with Phaeocystis sp. in McMurdoSound, Antarctica. Mar. EcoL Prog. Ser., 105, 179-189.
Sell,A.F. and OverbeckJ. (1992) Exudates: phytoplankton-bacterioplankton interactions in PluBsee. J.Plankton Res., 14, 1199-1215.
ShimetaJ. (1993) Diffusional encounter of submicrometer particles and small cells by suspension feeders.LimnoL Oceanogr., 38, 456-465.
Suttle.C.A. (1992) Inhibition of photosynthesis in phytoplankton by the submicron size fraction concentratedfrom seawater. Mar. EcoL Prog. Ser., 87, 105-112.
Suttle.C.A. and Chen,F. (1992) Mechanisms and rates of decay of marine viruses in seawater. AppL Environ.MicrobioL, 58, 3721-3729.
Suttle.C.A., ChanAM. and Cottrell,M.T. (1990) Infection of phytoplankton by viruses and reduction ofprimary productivity. Nature, 347, 467-469.
ToranzcA-E., BariaJ.L. and HetriclcJ.M. (1982) Antiviral activity of antibiotic-producing marine bacteria.Can. J. MicrobioL, 28, 231-238.
Turley.C.M., Newell,R.C. and RobinsJD.B. (1986) Survival strategies of two small ciliates and their role inregulating bacterial community structure under experimental conditions. Mar. EcoL Prog. Ser., 33, 59-70.
van BoekeLW.H.M., Hansen.F.C, Riegman.R. and Bak,R.P.M. (1992). Lysis-induced decline of aPhaeocystis spring bloom and coupling with the microbial food web. Mar. EcoL Prog. Ser., 81, 269—276.
van Ettenj.L., LarieJ-C. and Meints,R.H. (1991) Viruses and virus-like particles of eukaryotic algae. Microb.Rev., 55, 586-620.
Williams,P_J. Le B. (1984) Bacterial production in the marine food chain: the Emperor's new suit of clothes.In Fasham,M J.R. (ed.), Flows of Energy and Material in Marine Ecosystems: Theory and Practice.Plenum Press, New York, pp. 271-299.
Willisms.PJ. Le B. (1990) The importance of losses during microbial growth: commentary on thephysiology, measurement and ecology of the release of dissolve organic material. Mar. Microb. FoodWebs, 4, 175-206.
Woo^A.M. and van ValenJ-M. (1990) Paradox lost? On the release of energy-rich compounds byphytoplankton. Mar. Microb. Food Webs, 4, 103-116.
Zachary.A. (1976) Physiology and ecology of bacteriophages of die marine bacterium Beneckea natrigens:salinity. AppL Environ. MicrobioL, 31, 415-422.
Received on July IS, 1994; accepted on January 13, 1995
