Viruses Systems in Marine Planktonic

FEATURE

VIRUSES IN MARINE PLANKTONIC SYSTEMS

By Jed A. Fuhrman and Curtis A. Suttle

Introduction ing regular structures such as icosahedrons (with The last 10-15 years have seen major changes or without a "tail"), filaments, and types with vari- in our views of marine planktonic food webs, pri- able morphology types (Figs. 1 and 2). marily from the realization that prokaryotic mi- There are three basic viral life cycles, all of croorganisms and small eukaryotes are responsible which start with attachment of the virus to a for a significant fraction, often 50% or more, of specific site and injection of the viral nucleic acid the primary production and heterotrophic con- into the host cell (Fig. 3). In the lytic cycle, this sumption of organic matter in these systems nucleic acid "'commands" the cell to produce mul- (Williams, 1981: Azam et al., 1983: Stockner and tiple viral progeny (often tens to hundreds) that Antia, 1986: Fuhrman, 1992). However, it has are released into the environment when the host only been in the past few years that marine scien- cell lyses (bursts). Also common among prokary- tists have investigated the roles of viruses in eco- otes is a process called lysogeny, in which the in- • . . early results sug- logical processes. Although this research area is jected nucleic acid can become integrated into the only in its infancy, early results suggest that host's genome. This "provirus'" is reproduced gest that viruses may viruses may be important agents in the mortality along with the host DNA for many generations of marine microorganisms and in controlling their until the survival of the host is threatened, at be important agents in genetic compositions. This paper will summarize which time specific biochemical mechanisms can the mortality of marine the experiments and measurements that have led trigger the onset of the lytic cycle. Viruses capable to this suggestion as well as the conceptual frame- of lysogeny are called "'temperate," and they can microorganisms . . . work within which they are interpreted. be lysogenic in certain strains of hosts, and purely Viruses are fundamentally different from most lytic in others. Many bacterial isolates have been other biological entities in that they have no me- found to be lysogens, which means that they har- tabolism of their own and must rely on a host or- bor one or more proviruses that can be induced to ganism for any energy-requiring process, includ- become lytic. Among both prokaryotes and eu- ing reproduction. In many cases the host-virus karyotes, "chronic infections" can also occur relationship is very specific, with the viruses in- where viral progeny are produced and shed from fecting a single species or sometimes subspecies the host via budding or extrusion of filaments, or genus. Because most organisms seem to be sus- while host metabolism and reproduction proceed ceptible to viral infection, this suggests there are relatively unaltered. In metazoans viral infections millions of different kinds of viruses• They are are usually specific to certain tissues or organs. very small, usually from 20 to 250 nm in diameter The Presence and Abundance of Marine (hence they would be defined by most chemical Viruses oceanographers as "'dissolved organic matter"). They are composed of nucleic acid packaged in a Viruses it!fecting specific hosts protein coat, with some viruses having lipid pre- The existence of viruses in the marine environ- sent as well. The nucleic acid can be DNA or ment has been known for many years (e.g., Kriss RNA, either of which can be single- or double- and Rukina, 1947: Spencer, 1955, 1960), although stranded. There are several morphologies, includ- most of the early studies were concerned less with enumerating viruses than with characterizing isolates in terms of salinity and temperature effects, J.A. Fuhrman, Department of Biological Sciences, Univer- morphology, host range, etc. (e.g., Spencer, 1960: sity of Southern California, Los Angeles, CA 90089-0371, Hidaka, 1971: Hidaka and Fujimura, 1971; USA. C.A. Suttle, Marine Science Institute, The University of Texas at Austin, PO Box 1267, Port Aransas, TX 78373-1267, Zachary, 1976, 1978; Baross et al., 1978, see re- USA. view by BOrsheim, 1993). These studies focused

OCEANO(;RAPH'~'Vo]. 6, No. 2°1993 5] polyhedral virus, 130-135 nm in diameter, that caused lysis of a widely distributed photosynthetic Head flagellate, Micromonas pusilla. They tested 35 different species from five classes of algae and found that the virus was specific for M. pusilla. Despite the potential ecological significance of their observations and indications that the virus could achieve high concentrations in seawater, research beyond initial characterization of the virus life cycle (Waters and Chan, 1982) was not pursued. It was not until more than a decade later that it was shown that viruses infecting representatives from a number of important groups of phytoplankton including cyanobacteria, diatoms, a prasinophyte, and a cryptophyte could be readily isolated from seawater (Suttle et al. 1990). Concentrations of viruses which infect particu- © lar hosts can vary widely, ranging from less than a few viruses per liter of seawater to concentrations > 108 L ~. For example, Spencer (1960) and Moe- bus (1987, 1992b) most often found abundances < 104 L-' for phages that infected marine bacterial Fig. 1: General morphologies of viruses (primarily bacteriophages), modified cultures. However, in the Kiel Bight (a somewhat from Ackermann and DuBow (1978). Another common term.for the head is polluted embayment of the Baltic Sea), Ahrens "capsid." Typical head diameters are 20-200 nm, although some larger ones (1971) found up to 3.7 X 107 phage L ~ that in- are known. Of the types shown, the only ones readily recognized in electron fected a strain of Agrobacterium stellatum, and micrographs from seawater are those with polyhedral heads (see Fig.2). Fila- Moebus (1992b) reported counts near Helgoland, mentous ones, which are ve~ flexible and are represented by the long thin rec- Germany, sometimes as high as 1.5 X 107 phage tangle on the right, and irregularly shaped ones are not easily distinguished L-' infecting one host strain; the highest abun- from other filaments or similar sized "blobs" in seawater. dances tended to last only a few days. In the coastal waters of Texas the concentration of phages that infect two taxonomically uncharacter- on viruses infecting bacteria (bacteriophages or ized marine bacteria (PWH3a and LMG1) varies phages). If such studies can be used as a guide, temporally and spatially, and is different for the the potential for viral infection seems high, as two hosts. For example, titers of viruses infecting 10-50% of marine bacteria isolates have been re- PWH3a varied from <0.7 to 101 L '; whereas, in ported to be susceptible to infection by cultured the same sample viruses infecting LMG1 ranged phage (Hidaka, 1977). However, Moebus and between 12 and 3.97 × 10 ~ L ~ (Suttle et al., Nattkemper (1983), who studied phage sensitivity 1991). The high densities were coincident with a of 1,382 bacterial isolates and found that many toxic dinoflagellate bloom. In the coral reef envi- could be infected with culturable phages, sug- ronment near Key Largo, Florida, bacteriophages gested that this might be because bacteria that can infecting cultured Vibrio species were also spa- be isolated from seawater may be particularly sus- tially variable, generally reaching highest concen- ceptible to such phages. trations (>75L -') in the nearshore and inshore envi- Evidence of viral infections in photosynthetic ronments where salinities were lowest (ca. 28-30 marine organisms began to accumulate in the ppt) and decreasing offshore where salinities were 1970s with a number of observations showing the >34 ppt (Paul et al., 1993). However, comparably The great variability of presence of virus-like particles (VLPs) in cultures high phage concentrations were found at one off- and natural assemblages of phytoplankton (e.g., shore station. Concentrations of viruses that infect specific virus abun- Chapman and Lang, 1973; Pienaar, 1976; see also photosynthetic plankton are also extremely vari- dances probably largely reviews by Dodds, 1979, 1983; Van Etten et al., able. For instance, viruses that infect the photo- 1991). These studies, however, provided no evi- synthetic flagellate Micromonas pusilla range in reflects the variability of dence that there were viruses in seawater that titer from <20 L' in the central Gulf of Mexico to host abundances... could infect and kill phytoplankton, although even 4.6 x 10~ L-' in some coastal water samples (Cot- earlier studies (e.g., Safferman and Morris, 1963, trell and Suttle, 1991). The great variability of 1967) had demonstrated the presence of viruses specific virus abundances probably largely reflects that infected freshwater cyanobacteria (prokaryotic the variability of host abundances, because the phytoplankton). The first evidence of infective viruses are dependent on hosts for reproduction. "phycoviruses" in seawater was provided by Of viruses infecting specific hosts, those found in Mayer and Taylor (1979) who isolated a large the highest known concentrations are cyanophages

52 OCEANOGRAPHY'VoI, 6, No. 2-1993 E

Fig. 2: Transmission electron micrographs showing the morphological diversity of marine viruses. A-C), Natural virus communities from the Gulf of Mexico; D and F), Cyanophages (S-PWM4 & S-PWP1), which infect strains of Synechococcus spp., E) Virus (MPV-SP1), which infects the photosynthetic flagellate Micromonas pusilla. Scale bar equals 100 nm.

(viruses that infect cyanobacteria) infecting the 1991), and a virus has recently been isolated that genus Synechococcus. This is of ecological infects a marine heterotrophic nanoflagellate (D.R. significance because the genus Synechococcus is Garza and C.A. Suttle, unpublished observations). among the most important primary producers in Because a large number of viruses are known to the open sea, thought to account for a substantial infect crustaceans in aquaculture, it seems likely portion of the carbon fixation in the world's that crustacean zooplankton would be susceptible Almost nothing is oceans (Joint and Pomroy, 1983; Li et al., 1983). to viral pathogens. For example, at least six fatal known about the In coastal waters and along a transect in the nutri- viral diseases of penaeid shrimp are recognized ent-deplete waters of the western Gulf of Mexico (Sindermann, 1990). Almost nothing is known abundance or ecologi- cyanophages infecting a single Synechococcus about the abundance or ecological significance of cal significance of strain occurred at concentrations ranging from 103 viruses that infect zooplankton, and this remains a L' to >108 L' (Suttle et al., 1993; Suttle and potentially fruitful area for research. viruses that infect Chan, 1993). The concentration of viruses varied The large-scale distribution of marine bacterio- zooplankton . . . seasonally and was dependent upon the host strain phages infecting several cultured bacteria was stud- of Synechococcus that was screened. ied by Moebus and Nattkemper (1981). They found Viruses also infect members of the zooplank- that in the Atlantic between Europe and Bermuda, ton. A number of viruses are known to infect mi- broad geographic patterns of host sensitivity were crozooplankton, including rotifers (Comps et al., apparent, and phages isolated from widely sepa-

OC[-ANOGRAPHY°VoI.6, NO. 2"1993 53 VIRUS LIFE CYCLES

/ LYSOGENIC /

"Temperate" phage

.o°,.,°a°,,,o,.°,., v,..o J into host ~ _ J ~- ~ !

Host with viral nucleic acid integrated into I 1 , 1- I genome or as extrachromosomal element ~ S / ~,~|0 | | 0/~, Induction / normal division ~, Host makes Copies of viral / J al~ W ~ nucleic acid and coat proteins ( , Host makes copies of viral , ~- ~ ~- ...... ~ ~ (- c-3(. nucleic acid and coat proteins L__gL_~ "~k ~ ~:9 ~ . ~,--~ }¢J \ X I~ I~ I~l t,M I~/ Normaldivision continues unless induction occurs Host releases progeny viruses without (, ~ / ~ / • "~ lysmg - by budding or extrusion ~-J _ \ virusesselt-assemblo ¢ ~/ J ~, ~'* d.~ ~/ norrnaldivision ~ C ~ \ within host -- -- '~ t3 ~ - ~ ~ ~

Host lyses to release pgogeny viruses Host progeny continue to release viruses unless "cured"

Fig. 3: General plan of the three basic virus life cycles, diagramming bacterial infection. To date. most of the observed and discussed effects of infection in the sea are associated with the lytic cycle (or the lytic part of the lysogenic cycle). Lysogeny is obviously of interest and is now beginning to be studied in marine samples, especially because a well-known induction factor is UV light, which is present in surface waters. Chronic infection, which has relatively subtle effects, has yet to be studied in marine samples.

rated geographic areas were able to cause lysis of depths to 97 m (Suttle and Chan, 1993; C.A. Suttle, certain hosts. Phycoviruses infecting some eukary- unpublished observations). They were also found in otic phytoplankton are also extremely widespread. a single seawater sample screened from coastal Viruses infecting Micromonas pusilla have been New York. Infectious cyanophages were distributed found in every coastal seawater sample screened, with the highest concentrations in nearshore waters including samples from the coasts of New York, coincident with the distribution of phycoerythrin- Texas, California, and British Columbia (Cottrell containing Synechococcus. In surface waters of the and Suttle, 1991), as well as in the oligotrophic Gulf of Mexico, cyanophages infecting certain central Gulf of Mexico. Similarly, cyanophages in- strains of Synechococcus are typically at least an fecting Synechococcus spp. have been found in order of magnitude or more abundant than the total every seawater sample of hundreds screened from concentration of Synechococcus spp. (Suttle and the Gulf of Mexico (Suttle and Chan, 1993). They Chan, 1993). The numbers were also found to de- were found at all times of the year, at salinities crease rapidly below depths of 30 m (C.A. Suttle, ranging from 18-70 ppt and 12-30.4°C, and at unpublished observations).

54 OCEANOGRAPHY'VoI.6, No. 2,1993 Natural marine virus communities 1993; Fuhrman et al., 1993; Paul et al., 1993, reviewed by BCrsheim, 1993). Most of the recent Perhaps not surprisingly, estimates of viral counts have been typically 10" to 107 mL' in abundance in seawater based on the number of ocean surface waters, and up to -10" mL' in the viruses infecting a specific host in culture greatly richest coastal waters, estuaries, and embayments. underestimate the total concentration of viruses Virus abundances >105 mL' have even been re- determined by microscopy. This is because >99% ported for the Southern Ocean in winter, where of bacteria in typical native heterotrophic bacterial phytoplankton and bacterial abundances are ex- communities are nonculturable on standard media tremely low (Smith et al., 1993). Most counts (Ferguson et al., 1984; Lee and Fuhrman, 1991): have been done by TEM, but counts using hence, bacteria used as bioassay organisms are epifluorescence microscopy yield similar results probably not representative of the natural micro- (Suttle et al., 1990: Hara et al., 1991: Proctor and bial community. Additionally, even minor differ- Fuhrman, 1992: Fuhrman et al., 1993). However, ences between host strains can sometimes affect counts by TEM have sometimes greatly exceeded susceptibility to phage infection (Ackermann and those by epifluorescence (Paul et al., 1991). DuBow, 1987), resulting in a minimum estimate Methodological details (filter and stain types, of phage abundance for a particular species. Fur- prefiltration, optics of the microscope, detection thermore, viruses that are noninfective can be by eye, film, or video) are probably critical. counted by microscopy but not with a bioassay, Counting methods are reviewed in detail else- which also must be borne in mind when interpret- where (Suttle, 1993). Viral abundance typi- ing and comparing results. Viral abundance typically parallels that of bac- Transmission electron microscopy (TEM) has teria, with abundances decreasing from nearshore cally parallels that of most often been the choice for viewing viruses di- to offshore waters and from surface to deep wa- rectly because it affords resolution of up to a few bacteria . . . ters: the number of viruses generally exceeds that nanometers, permitting visualization of the shape of bacteria by ~5- to 10-fold (Hara et al., 1991, and morphological details of the viruses. Sieburth Paul et al., 1991, 1993; Wommack et al., 1992, (1979) used TEM to observe viruses in seawater Cochlan et al., 1993). The strength of this rela- but did not provide concentration estimates. The tionship, as well as the fact that bacteria are by far first quantitative estimates of viral abundance in the most abundant potential hosts, indirectly sug- seawater were provided by Torrella and Morita gest that the majority of viruses infect bacteria (1979) who reported >104 viruses mL' in coastal (Paul et al., 1991; Cochlan et al., 1993). However, Oregon waters. These were obvious underesti- the data do not exclude the possibility that viruses mates because only viruses retained by 0.2 #m infecting other organisms may be numerically and pore size filters were counted. These authors sug- ecologically important members of marine viral gested that the viruses were primarily bacterio- communities. It is important to keep in mind that phages, and that processes such as genetic transfer cyanophages (e.g., Suttle and Chan, 1993) and could occur in the sea via viral infection. Once even some viruses that infect eukaryotic phyto- again, these exciting observations were not pur- plankton (Van Etten et al., 1991) can be morpho- sued in a rigorous or systematic fashion by other logically indistinguishable from phages which in- investigators lbr several years. fect heterotrophic bacteria. Consequently, it is It was not until the late 1980s that direct counts premature to draw any firm conclusions about of viral abundances in the range of 10~ mL ' were what kinds of viruses dominate natural systems. reported by Sieburth et al. (1988) using epifluorescence microscopy to count virus-sized particles Diversity of Viral Communities stained with a DNA-specific fluorochrome The morphological diversity within natural ma- (DAPI), and by Proctor et al. (1988) using TEM. rine virus communities is enormous (Fig. 2, see These were followed shortly by estimates ranging also size data reviewed by BCrsheim, 1993), and from <107 to >10 ~ mL ' for a variety of marine some extremely large marine virus-like particles waters (Bergh et al., 1989; Proctor and Fuhrman, have been found with heads the size of small bac- 1990), including the Gulf Stream and open ocean. teria (0.4 #m) and 2.5 #m-long tails (Bratbak et Interestingly, new technology was not required for al., 1992a). This diversity suggests that they are the "discovery" of high virus concentrations in also diverse in terms of the hosts that they infect. seawater: rather, old methods (e.g., Sharp, 1949) In fact, because there are ~ 107 viruses mL ' in were simply applied to a new problem. mesotrophic seawater, but generally only a few Subsequently, high abundances of marine hundred viruses mL ~ or less infecting a particular viruses were confirmed in many samples from host (with the exception of cyanophages), it has several locations and depths, using a variety of been suggested that the viruses in a given milli- methods (B0rsheim et al., 1990; Bratbak et al., liter of seawater could infect >10 ~ different host 1990; Suttle et al., 1990, Hara et al., 1991, Heldal species (Suttle et al., 1991)! However, this sug- and Bratbak, 199l; Paul et al., 1991; Bratbak et gestion should be tempered by the possibilities al., 1992b; Wommack et al., 1992; Cochlan et al., that most of the virus particles in seawater are not

OCEANOGRAPHY°VoI. 6, No. 2o1993 55 infective (see below) or that the model virus-host Among the few early studies on decay rates of systems that have been isolated are not representa- native viruses in aquatic environments were those tive of natural virus communities. of Zachary (1976), who found that phages infect- The genetic diversity of marine viruses is cer- ing the estuarine bacterium Vibrio natriegens de- tainly greater than the observed morphological di- cayed relatively slowly in estuarine water. One versity. For example, a group of morphologically isolate (nt-6) decayed at 7-18% per day for 60 d; indistinguishable viruses that infect the photosyn- whereas another (nt-1) decayed at 18% per day for thetic flagellate Micromonas pusilla are geneti- the first 10 days, but only 0.3-0.7% per day for cally very different, even though some were iso- the next 50 days. This change in the decay rate lated from the same water sample (Cottrell and was possibly because the phage found a refuge Suttle, 1991 ). from the inactivation factor(s) or the inactivation Dynamics of Viruses and Viral Communities factor was reduced considerably. In contrast, the phages showed no inactivation over _~ 60 d in au- Viruses are a dynamic component of marine systems. Rapid changes in viral abundance have toclaved or filter-sterilized estuarine water, or ster- been observed in microcosms filled with coastal ile saline media. The data suggested that inactiva- seawater in which certain size classes of viruses tion was heat-labile and particle-associated (e,g., increased exponentially at 0.4 d -~ for 5-7 d (Bcr- microbial), and that different phages can decay at sheim et al., 1990), and during a diatom bloom different rates in the same water. More recently, when viral abundance increased from 5 x l0 s to Moebus (1992a) found comparable rates (typical 1.3 x 107 mL ' at the bloom's peak before declin- inactivation rates of 10-30% per day) and high ing at a net rate of 0.33 d-' (Bratbak et al., 1990). variability in a study of survivability of nine cul- These authors suggested that the rapid increases tured phages suspended in water sampled from may have resulted from induction by light of near Helgoland in the North Sea. He concluded that indigenous bacteria have the predominant role • . . viruses are being prophages within lysogenic bacteria (further discussed by Thingstad et al., 1993). Similarly, viral in phage inactivation. produced and abundance changed as much as twofold in 24 h in Given the potential importance of attachment to destroyed at rapid but 60-L enclosures of Norwegian coastal water (Hel- particles as a mechanism for removal of viruses, dal and Bratbak, 1991). Such dynamics indicate Murray and Jackson (1992) constructed a model variable rates. that viruses are being produced and destroyed at investigating the potential effect of single-celled rapid but variable rates. organisms and other particulates on the removal There has long been interest in determining rates of viruses from seawater. They concluded loss rates and mechanisms of viral decay in sea- that there was a fairly close relationship between water. Potential decay mechanisms include extra- the abundance of bacteria and the removal rates of cellular digestion by enzymes (free in the water viruses in seawater. They postulated that virus at- or on the surfaces of cells), damage from UV or tachment to nonhost organisms is potentially a visible light, adsorption onto living or nonliving major source of virus removal in seawater. particles and possible subsequent ingestion of the Recently, decay of infectivity of three cultured particle by grazers, unsuccessful infection of a marine phages containing double stranded DNA host, direct ingestion by protists, and attack by was estimated from Texas coastal water at 22 to chemicals other than enzymes. Most viral decay 67% per day in the dark, with different viruses de- studies have focused on the decay of infectivity caying at different rates (Suttle et al., 1992; Suttle of human pathogens and coliphages (phages in- and Chen, 1992). Again, interaction with heat la- fecting human intestinal bacteria). Data from such bile particles was responsible for much of the studies suggest that infectivity decays quickly decay because viruses did not decay or decayed with 90% of the infectious bacteriophage isolates very slowly in autoclaved, 0.2-ttm-filtered or ul- typically destroyed in 1-10 days (Kapuscinski tracentrifuged seawater. These interactions could and Mitchell, 1980); average daily decay rates are include passive adsorption in addition to enzy- ~20-90% per day. The data from these earlier matic microbial processes. Cyanide, an inhibitor studies suggested that decay is primarily biologi- of respiration, caused rates to decrease by ~60%, cally mediated, although chemical effects and ef- indicating that energy-requiting biological processes fects from solar radiation were also implicated were responsible for some of the decay. As decay (e.g., Mitchell, 1971; Shuval et al., 1971; Berry rates were greatly reduced in 0.8- or 1.0-#m- and Noton, 1976). In addition, colloids and par- filtered seawater, whereas bacteria numbers were ticulate material were shown to adsorb and re-re- not, it suggested that the largest bacteria (removed lease coliphages in seawater, effectively slowing by the filtration) or other organisms were respon- decay (Bitton and Mitchell, 1974; Bitton, 1975; sible for some of the decay, or that filtration inhib- Gerba and Schaiberger, 1975; Smith et al., 1978). ited bacteria-mediated decay. Decay rates were These studies identified mechanisms that could also accelerated when viruses were added to cul- be important in regulating the abundance of ma- tures of heterotrophic nanoflagellates, correspond- rine viruses. ing to 3.3 viruses inactivated flagellate ~ h '. These

56 OCEANOGRAPHY'VoI. 6. No. 2"1993 results closely agree with observations that free viruses have no metabolism so they cannot phagotrophic nanoflagellates ingest and digest repair themselves), or to the ability of virus-en- fluorescently labelled marine viruses (Gonzalez coded nucleic acid polymerases to function on ra- and Suttle, 1993). In the absence of solar radia- diation-damaged DNA. tion, a major factor responsible for viral decay The observed rapid viral dynamics imply actual was probably attachment to fragile, heat-labile ag- disappearance of the viruses, not just loss of infec- gregates. However, in the presence of full sunlight tivity. Heldal and Bratbak (1991) estimated disap- decay rates were greatly accelerated (0.4-0.8 h-~). pearance rates in field samples by attempting to Even when UV-B was blocked, rates were as high stop virus production with cyanide while deter- as 0.17 h '. mining removal rates of the remaining viruses The data described above were the basis of a from TEM counts. Cyanide will stop virus pro- model that partitioned viral decay among different duction by inhibiting host respiration, without di- mechanisms in the top 10 m of a hypothetical rectly affecting enzymes (e.g., proteases or nucle- coastal water column (Suttle and Chen, 1992). ases) that might destroy viruses• In contrast to The average decay rate of infectivity attributable culture results (see above), cyanide was not found to micrometer-scale particles (adsorption plus mi- to affect viral decay rates in the sample that was crobially mediated decay) was 0.41 d ', and that tested. Using this approach they measured ex- resulting from solar radiation was 0.38 d '. At typ- tremely rapid decay rates of 0.3 to 1.1 h ~ for ical viral and flagellate abundances, grazing by slightly over 60% of the viral population during nanoflagellates was of minor importance, account- the first few hours. However, ~40% of the ing for a loss rate of only 0.002 d ~. Extrapolating viruses were relatively persistent after this initial to a 200 m oceanic water column, light could be rapid decline and disappeared at much slower the major factor causing decay of viral infectivity. rates (<0.05 h '). Furthermore, as sunlight destroys infectivity ap- An important contribution of these studies was proximately 15 times more rapidly than it does the idea that production rates can be estimated by virus particles (Suttle et al., 1993), a large propor- comparing loss rates in cyanide treated and un- tion of the viruses in seawater may not be infec- treated samples (where production is continuing); tive. This should also lead to a strong diel signal the difference between the two represents virus in the concentration of infectious viruses. Even in production. They estimated the total bacterial unlit waters many of the viruses may be noninfec- lysis rate from phage infection by assuming that tive as viral inactivation (by all mechanisms) the virus community size is maintained at a con- likely occurs faster than removal. stant level (as they observed), each lysis event re- The model presented by Suttle and Chen leases 50-100 progeny viruses ("burst size" esti- (1992) was based on three randomly selected ma- mated in their study), and 50% of the viral rine virus isolates, which showed a range of sensi- population decays at the faster measured rates. tivities to solar radiation. Other marine viruses are This model yielded estimates of 2-24% of the less susceptible to sunlight. We recently con- bacterial population being lysed per hour, thus re- ducted tests on the same day at Los Angeles, Cali- quiring bacterial growth rates of 0.4-6 d ~ to bal- fornia, and Port Aransas, Texas, using local sea- ance virus-induced mortality. The higher end of water and ambient sunlight to test the effect of this range is much greater than most growth rate solar radiation on a double-stranded DNA bacte- estimates for marine bacteria in natural waters riophage isolated from the North Sea (H40/I; (0.5-2 d-~), so these results have met with some Frank and Moebus, 1987). Photosynthetically ac- skepticism. More recently, Bratbak et al. (1992b) tive irradiance at Port Aransas during the 7-h in- found slower decay rates (0.3-0.4 h ') for --30% cubation averaged 1,172 #mol photons m-" s -~. of the virus population (the rest decaying more Decay rates of H40/1 were relatively slow in full slowly) in Danish coastal waters• Using an esti- sunlight, ranging from 0.05-0.11 h ' at both loca- mate of 100 for burst size, this corresponded to a tions. Another phage isolated from the North Sea more believable 72% per day bacterial mortality. • . • susceptibility of (H85/1) showed similar decay rates in Los Ange- Yet, the measured bacterial production rate (from les, but in contrast, one of the viruses used in pre- thymidine incorporation) was capable of support- infectivity to sunlight is vious sunlight decay experiments (PWH3a-PI) de- ing only one sixth of the estimated viral produc- virus dependent . . . cayed nearly three times as fast (0.32 h ~) at Port tion rate. Moreover, these authors estimated that Aransas. Thus the susceptibility of infectivity to grazing by protists consumed bacteria about twice sunlight is virus dependent, and the model de- as fast as the estimated bacterial production rate. scribed above may overestimate or underestimate Obviously, the rate measurements cannot all be the actual decay rates experienced by individual correct. Although the use of a higher thymidine components of natural marine viral communities conversion factor would help, factors in the com- when they are exposed to solar radiation. The rea- monly accepted range would not be adequate to sons for the variation in light susceptibility are un- account for the sevenfold increase in production known, but may be related to differences in the that would be necessary to balance all the mea- DNA repair mechanisms of the hosts (note that surements.

OCEANOGRAPtIY'VoI.6, No. 2°1993 57 It is also difficult to reconcile some of the very see discussion above). In addition, Suttle and fast estimates of viral decay based on particle dis- Chen (1992) estimated that 4-13% of the bacterial appearance with the much slower rates based on community would have to be infected daily to decay of infectivity, because decreases in infectiv- support the observed viral decay rates. If bacteria ity should provide more conservative estimates of in this area typically double about once per day, viral decay than the disappearance of viruses. It is and if it is assumed that half of the bacteria are re- possible that the cultured viruses in infectivity moved each day to maintain steady state, then studies are not representative of those in natural viruses are responsible for 8-26% of the bacterial communities. The following are alternative expla- mortality. nations: 1) very rapid decay rates may be charac- Other evidence that viruses are important teristic of certain water types, 2) the cyanide pro- agents of mortality for bacteria and phytoplankton tocol overestimates virus disappearance rates, or was obtained by adding virus-sized material, con- 3) decay rates of infectivity have been inhibited in centrated from seawater by ultrafiltration, to sea- the other studies. Recent cyanide experiments in water samples, and documenting the effects on California waters have sometimes yielded the un- bacteria and phytoplankton communities. expected (and uninterpretable) result of a Significant declines in bacteria abundances (Proc- significant increase in viral abundance after the tor et al., 1988; Proctor and Fuhrman, 1992) and addition of cyanide, indicating that unknown phytoplankton photosynthetic rates and biomass processes can occur in these experiments and cau- (Suttle et al., 1990; Suttle, 1992: Peduzzi and tion must be used in their interpretation (J.A. Weinbauer, 1993) occurred in samples exposed to Fuhrman, R.T. Wilcox and R.M. Noble, unpub- these concentrates. The effect was eliminated if lished observations). Clearly, studies need to be the concentrates were heat-treated before addition. done in which losses of virus particles and de- Photosynthetic rates declined within minutes of • . . only a subset of creases in infectivity are monitored simultane- adding the concentrates and were reduced by as ously. much as 78%. In fact, as little as a 20% increase the phytoplankton A recent contribution that may help settle some in the 2-200 nm size fraction reduced photosyn- community was of this controversy is an approach for directly thetic rates by as much as 50%. Microautoradiog- measuring virus production in natural samples raphy indicated that only a subset of the phyto- affected... using radioactive phosphate or thymidine (Steward plankton community was affected and that et al., 1993a,b). The isotopes are incorporated into phytoplankton >3 #m were inhibited the most. viral nucleic acids, which are purified by a combi- Note that the rapid response of photosynthesis nation of physical separation and chemical or en- cannot be from cell lysis, which probably occurs zymatic treatments. By this method they found several hours after infection, and more likely rep- that virus production had a strong onshore-off- resents regulation of cellular processes• Moreover, shore gradient in the waters off Southern Califor- only a subset of the phytoplankton community nia. Production rates ranged from <1 X l0 s (unde- was affected and photosynthetic biomass increased tectable) to 2.3 x 10" viruses L ~ d-', with values after a lag of >1 d (Suttle, 1992; Peduzzi and highest in a coastal lagoon and lowest in offshore Weinbauer, 1993). Although these studies indi- oligotrophic waters. The values are conservative cated that the viral size fraction is strongly hioac- because they do not account for losses during tive, the possibility cannot be dismissed that other filtration or purification, which can be >50%. substances (e.g., proteins) were concentrated by the procedures, and contributed to the effect, Viruses as a Cause of Microbial Mortality Independent documentation of the importance It is important to understand the mechanisms of viruses was obtained from observing thin sec- responsible for the mortality of marine bacteria tions of natural plankton communities with TEM and phytoplankton, as different routes of microbial (Proctor and Fuhrman, 1990). Culture studies have mortality yield different pathways of organic mat- shown that assembled (mature) phage typically ter flow and oxidation in the ecosystem (discussed appear at the final stage of the infection cycle; below). Traditionally, flagellates and ciliates have hence the proportion of infected cells can be esti- been regarded as the only important agents of bac- mated from the proportion of bacteria and terial loss (reviewed by McManus and Fuhrman, cyanobacteria containing mature phage. In rich 1988, and Pace, 1988); however, attempts to bud- coastal, as well as oligotrophic offshore Atlantic get bacterial production and removal by protozoan environments, ~ 1-4% of the bacteria and 1-3% grazers have often led to imbalances, suggestive of the cyanobacteria were visibly infected (3 or of unknown mechanisms for the loss of bacteria more phage per cell). These values were converted (Servais et al., 1985; McManus and Fuhrman, to mortality estimates using a three-part steady- 1988; Pace, 1988; Sherr et al., 1989). state model. The first model component was that Calculations of extremely high viral loss rates intracellular mature phage are only visible for imply that high rates of bacterial mortality due to about the last 10% of the infection cycle (from viral lysis must occur to support viral production limited literature data); hence, the percent of visi- (Heldal and Bratbak, 1991; Bratbak et al., 1992b, bly infected cells must be multiplied by about 10

58 OCEANOGRAPHY°VoI. 6, No. 2o1993 to estimate the total infected cells. Second, litera- A similarly low percentage has been calculated by ture data indicated that the time between cell in- J.B. Waterbury (unpublished observations), based fection and lysis (latent period) is approximately on abundances and diffusion rates of cyanobacte- the same as the generation time of the uninfected ria and cyanophages. host grown in the same medium. Thus an infected Another method that has been used to estimate cell is likely to die within a generation. Third, at the fraction of bacteria containing mature viruses steady state, each cell division results in one cell has been to treat samples with streptomycin to that will live and one that will die within a gener- lyse infected ceils and release the viruses, which ation time (on average, to maintain steady state), are observed by TEM (Heldal and Bratbak, 1991; so any factor that leads to the death of 50% of the Bratbak et al., 1992b). These authors report that cells in a generation is responsible for 100% of the percent of bacterial cells containing mature the mortality. This model suggested that one must phage particles was 8-14% in coastal Norwegian multiply the percent of cells visibly infected by a waters, and 12-29% in coastal Danish waters, "conversion factor" of 20 to estimate the percent with the latter showing a diel variation with low- of total mortality due to viral infection. It was est values near midnight and highest in afternoon. concluded that as much as 30% of the total These percentages are much higher than estimated cyanobacterial mortality (from all causes) and from thin sections by Proctor and Fuhrman 60% of the bacterial mortality could be due to (1990), and a conversion factor of 10-20 to esti- lysis by phage. These estimates were a first ap- mate the percentage of bacterial mortality from proximation based on an uncertain conversion fac- the percentage of infected cells cannot be applied tor, and do not take into account the possibility to these data. It is important to realize that the that an infected bacterium may be eaten by a pro- 10-20 conversion factor is empirical and applies tist before the cell lyses. only to thin sections, so this conversion would not These studies were extended to microorganisms be expected to apply to other means of observing in sinking particulate material collected from sedi- infection. The total number of cells with mature ment traps at 30-400 m in the North Pacific (Proc- phage will be higher than that observed in thin tor and Fuhrman, 1991). Viruses were common in sections because thin sections observe only a por- this material and appeared aggregated. The fraction of each cell, and the viruses can be located tion of cells containing mature phage was elsewhere in the cell. Also, Proctor and Fuhrman 0.7-3.7%. The model was further refined for this (1990) counted only cells with at least three visi- study, and approximate lower and upper bounds ble mature viruses per section (to indicate the cell were applied (still from limited literature data). was in a late stage of infection), and many cells The conclusion was that 2-37% of the cells in the had fewer apparent viruses. While there are no es- sedimenting matter were likely to be killed by timates of mortality from the streptomycin lysis. method, the observation that a minimum of Recently, the mortality model parameters were 8-29% of the bacteria were infected by viruses investigated in culture using two species of phage- suggests that viruses have a significant impact on infected heterotrophic marine bacteria (Proctor, these systems. • . . mortality from 1991; Proctor et al. 1993). Almost all of the visi- The virus production data from Steward et al. ble intracellular phage accumulated within the last (1993b) suggest mortality from viral infection is viral infection is likely 25% of the infection cycle, with most appearing likely to be most important in coastal embayments to be most important within the last 10%. Also, the averaged latent pe- as opposed to open ocean waters. As with the Hel- riods were very close to the generation times of dal and Bratbak approach, it is necessary to know in coastal embay- uninfected hosts, but there was considerable varia- the burst size to estimate mortality rates from tion around the mean. This basically confirmed the virus production measurements. Steward et al. ments... earlier interpretation and implied that the best cur- used a large range of burst sizes (10-300), be- rent estimate for the "conversion factor" is because of uncertainty with this number (the lowest tween 10 and 20, assuming that these laboratory burst sizes lead to the highest mortality estimates). model systems are representative of native marine The estimated fraction of bacterial mortality at- phages. The field results thus indicate that perhaps tributable to viruses was 25-740% in Pefiasquitos 6-60% or more of the total bacterial mortality is Lagoon near San Diego, 12-372% in Mission due to viral infection. It should be noted that the Bay, and 1-40% in offshore and nearshore Pacific same factor probably does not apply to cyanobac- Ocean samples. Estimates from Pacific Ocean teria. Padan and Shilo (1973) and Leach et al. samples overlap with those of Proctor and (1980) indicate that mature intracellular cy- Fuhrman (1990), but the range is too large to say anophage can become abundant about halfway that there is "agreement." through the infection cycle, suggesting a conversion factor of about 4. Thus given that only Effects on the Diversity of Microbial 1-3% of the native population appear infected Communities (Proctor and Fuhrman, 1990), viruses may account One of the most important roles of viruses in for only 5-10% of the mortality of cyanobacteria. planktonic communities is likely the maintenance

OCI~2ANOGRAPI1Y'Vo1. 0, NO. 2"1993 59 of diversity. More than 30 y ago G.E. Hutchinson phagotrophic protists developed. They suggested (1961) focused attention on a question that had that the viruses primarily influenced the genetic plagued ecologists for years. Why do so many structure of the algal community (species and/or species of phytoplankton with similar resource re- strain composition) rather than its size. A similar quirements coexist in relatively homogeneous conclusion was reached by J.B. Waterbury (un- bodies of water, when theory would predict that a published observations) regarding the marine few species should be competitively dominant? cyanobacterium Synechococcus, and this generally Although the question was posed for phytoplank- follows the conclusions of Lenski (1988) based on ton, given our recent appreciation of the diversity extensive work with bacterial cultures. However, of aquatic bacteria and protozoa it is appropriate the most convincing evidence that viruses may to extend the question to these groups. Obviously, terminate phytoplankton blooms comes from many factors contribute to the maintenance of mesocosm studies in which the collapse of Emilia- community diversity including patchiness, non- nia huxleyi blooms was associated with the ap- steady state conditions, selective predation, differ- pearance of large polyhedral virus-like particles ences in the relative amounts of resources re- inside the cells and high concentrations of mor- • . . viral infection is quired, allelopathy, etc., but viral infection is phologically similar particles in the surrounding potentially one of the potentially one of the most important mechanisms seawater (Dundas et al., 1992; Bratbak et al., maintaining diversity (Suttle et al., 1990, 1992; 1993). It seems likely that although viruses may most important mech- Fuhrman, 1992: Thingstad et al., 1993). The rate not be responsible for the termination of all or at which a viral infection will spread is very de- even most algal blooms, they play important roles anisms maintaining pendent on the frequency with which viral parti- in regulating phytoplankton species compositions diversity. cles will encounter a suitable host. Since infective and dynamics. viruses may have a relatively short residence time in seawater (see above) rapid propagation of a Viruses and Organic Matter Cycling viral infection will occur best at relatively high From a food web point of view, the fate of host densities (>104 host cells mL t, Wiggins and viral "biomass" is important. Individual infected Alexander, 1985). Still, infections can propagate cells lyse and release many viruses plus extensive slowly with host and phage densities of even 10 cell debris. In a long-term, near-steady state situa- mL -~ (Kokjohn et al. 1991). Although the variety tion, one would expect that an average of only one of hosts for marine viruses can be complex, virus from each burst survives to infect another viruses infecting marine bacteria and phytoplank- host successfully; otherwise, the virus abundance ton typically infect members of a single species or would explode or decline, neither of which seems genus (e.g., Mayer and Taylor, 1979; Suttle and to have happened for the group as a whole (this is Chan, 1993). Consequently, even in the presence for virulent infections; with lysogeny, even fewer of very low viral titers, a high concentration of a viruses probably survive to infect a new host, be- susceptible host will lead to rapid viral propaga- cause these types have an alternate survival strat- tion and lysis of the host population. The host is egy). Bratbak et al. (1990), Proctor and Fuhrman able to survive because there are invariably some (1991), and Fuhrman (1992) present food web cells present that are resistant to infection by the models that describe the effects of virus-caused viral pathogen, and the extent of host survival will mortality on the microbial food web. These have depend on their genetic diversity, as well as the generally assumed that the cell debris from lysis abilities of the viruses to co-adapt (see review by and the material in released viruses return to Lenski, 1988)• The result is that species that "feed" the bacteria, in a closed loop that essen- would otherwise be the competitive dominants tially oxidizes C and regenerates N and P. A may be kept in check by the presence of viral quantitative steady-state model presented by pathogens. Rapid propagation of viral infections Fuhrman (1992) compared two microbial systems, when host densities are high may be the prime one having all bacterial mortality from protistan reason why planktonic communities are not domi- grazing, and the other having 50% of the mortality nated by a few species in nature. from viruses and 50% from protists. The material Until recently, there has been little direct evi- released from lysed bacteria was assumed to cycle dence for the termination of phytoplankton back to the bacteria as dissolved organic matter blooms by viruses in nature, although reports of (DOM). The inclusion of viruses caused a 27% in- bloom lysis (e.g., Gieskes and Elbrachter, 1986) crease in both bacterial secondary production and and difficulties in balancing rates of primary pro- bacterial respiration, a 37% loss in the export of ductivity with loss rates for phytoplankton (e.g., bacterial carbon to nanozooplankton grazers, a Walsh, 1983) suggested that the process may be 25% loss in nanozooplankton secondary produc- important. In contrast, Sieburth et al. (1988) sug- tion, a 13% loss in microzooplankton production, gested that even though virus-infected cells were and a 7% loss in macrozooplankton production. visible throughout a bloom of the small chryso- Thus the viruses caused a shift in food web activ- phyte Aureococcus anophagefferens, the bloom ity (most notably, production and respiration) declined only after significant numbers of from larger organisms to bacteria, with the some-

60 OCEANOGRAPHY'VoI• 6, No. 2-1993 what unexpected conclusion that even in the ab- planktonic communities and on the large scale sence of increased primary production, the pres- flow of carbon, energy, and nutrients, which will ence of active viruses can lead to significant in- allow us to incorporate viruses and viral-mediated creases in bacterial production and respiration processes into quantitative marine ecosystem rates. However, these increases were accompanied models. by less rather than more carbon transfer to higher trophic levels. Modification of the model to in- Acknowledgments clude infection of primary producers (10% of phy- The authors thank W. Cochlan, G. Steward, F. toplankton mortality), and consumption of viruses Azam, J. Wikner, and D. Smith for sharing unpub- by nanozooplankton (13% of virus production) in- lished results, and K. Moebus for supplying bacte- creased bacterial production and respiration rates riophage isolates from the North Sea. R.T. Noble, even more (33% above the level without viruses), R. Wilcox, and D.R. Garza were responsible for while at the same time slightly moderating the de- the virus survival experiments. A.M. Chan and cline in nanozooplankton production (20% loss in- M.T. Cottrell supplied the electron micrographs. stead of 25%; J.A. Fuhrman, unpublished observa- J.B. Cottner and two anonymous reviewers made tions). useful suggestions. The effect of viruses on carbon and nutrient flow has particular bearing on the question of References whether bacteria are a "link or sink" in the food Ackermann, H.-W. and M.S. DuBow, 1987: Viruses of Prokaryotes. In: General Properties of Bacteriophages, web. 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Variations in the relative significance rio parahaemolyticus bacteriophages and other Vibrio bacteriophages in marine samples. Appl. Environ. Mi- sinks for C . . . of viruses may help explain why Ducklow et al. crobiol., 36, 492-499. (1986) found the bacteria to be a sink while Sherr Bergh, 0., K.Y. Bcrsheim. G. Bratbak and M. Heldal, 1989: et al. (1987) indicated they can be more of a link. High abundance of viruses found in aquatic environ- An additional effect of viruses on nutrient cy- ments. Nature, 340, 467-468. cling involves their roles in marine aggregates, Berry, S.A. and B.G. Noton, 1976: Survival of bacteriophages in seawater. Water Res., 10, 323-327. which are thought to be major agents of transport Bitton, G•, 1975: Adsorption of viruses onto surfaces in soil of matter to the deep sea. As stated above, Proctor and water. Water Res., 9, 473-484. and Fuhrman (1991) noted significant viral infec- Bitton, G. and R. 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