INTRODUCTION

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From studies of the genetics and biology of viruses has come a more profound understanding of the basic biological processes of life, not the least of which has been the discovery of DNA as the carrier molecule of genetic information (125) and mRNA as an intermediate molecule in the transfer of genetic information to the ribosomes (43). Other breakthroughs in molecular biology attributable to bacteriophage models are the definition and mapping of the first gene (18); discovery of the discontinuous nature of DNA replication (222); discovery of restriction endonucleases (212); and the mechanics of gene regulation (261). Indeed, basic research on the biology of the bacteriophage has been fundamental to the establishment of the field of molecular biology (73). The value of basic research to technological and economic advancement is perhaps best illustrated by the historical link between basic bacteriophage biology and the present-day, multibillion dollar biotechnology industry.

In contrast to extensive information on the biology and genetics of viruses, there is only a limited understanding of the occurrence and distribution of viruses in microbial ecosystems and in situ relationships between viral and host communities in the natural environment. The lack of recognition of viruses as naturally occurring organisms was most notably exposed with the discovery that viruses are abundant in a variety of aquatic environments, often exceeding bacterial abundance by an order of magnitude (19, 257). It was a surprise to learn from direct transmission electron microscope examination of marine virio- and bacterioplankton the astounding abundance of virus-like particles in seawater, considering that marine bacteriophages were first described in detail more than 35 years ago (301, 302, 331, 332). As an example, a transmission electron micrograph of unfiltered Chesapeake Bay water is shown in Fig. 1. The realization that in most aquatic environments the virioplankton is the most abundant plankton class has revived scientific investigation into the natural state of viruses in aquatic environments. Important questions raised by discovery of the abundance of viruses in natural ecosystems challenge accepted views of the aquatic microbial food web and the hypothesized singular role of obligate parasites in controlling microorganism population abundance and diversity.

View larger version (173K): [in this window] [in a new window]   FIG. 1.   Transmission electron micrograph of an unfiltered Chesapeake Bay water sample (magnification, ca. ×36,000). a, short-tailed or nontailed virus-like particle; b, tailed virus-like particle; c, bacterium, coccal morphotype; d, bacterium, vibrio morphotype.

This review is divided into four sections. The first and second sections cover methods for viral direct counting, reports of naturally occurring virioplankton abundance in a variety of aquatic environments, and the correlation between changes in virus abundance and changes in other important ecological parameters. The third examines aspects of aquatic bacteriophage biology which have a significant influence on host infection in aquatic environments. The final section focuses on viral infection and lysis, as both a factor in the mortality of host populations and a mechanism influencing genetic and clonal diversity of host populations. In general, the discussion is focused on in situ measurement of virioplankton populations. Model phage-host systems are included, as appropriate, for background information. Readers interested in aquatic phage-host relationships will find relevant information in reviews by Børsheim (29) and Proctor (253) and an earlier review by Moebus (193). A brief synopsis of current views on marine virus ecology was recently provided by Furhman (91). The distribution and survival of human and animal pathogenic enteroviruses in aquatic environments are not covered herein, since detection and distribution of human disease-causing viruses in natural waters have been extensively reviewed elsewhere (99, 103, 104, 112, 182).

ENUMERATION OF VIRUSES IN WATER SAMPLES

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Introduction

Discovery of the abundance of viruses in natural waters reflects the development of direct counting methods for bacterial enumeration. From the use of direct-counting methods to enumerate bacteria in environmental samples (127, 381), it has been found that viable counts, obtained using culture methods, significantly underestimate the number of bacteria in the sample. This finding is not surprising since it was suspected from the early days of bacteriology, when staining methods were used to enumerate bacteria. Newer techniques yielded results that led many investigators to reject conclusions regarding the ecology of bacteria obtained solely by culture. Molecular methods developed for analyzing bacterial population dynamics and diversity have revealed large populations of unculturable bacteria in the environment, further fueling speculation that bacterial diversity may be 100 to 1,000 times greater than that suggested by results of studies involving culture methods (60).

Indirect titer determination by plaque assay (4), coupled with the most-probable-number method (150, 309), has routinely been used to enumerate viruses in water samples but has only recently been used to elucidate the ecology of viruses. For example, the abundance and distribution of coliphages in natural water samples have been determined by a plaque assay (99, 118, 244). In general, estimates of the abundance of specific viruses by culture methods have been so low that preconcentration of viruses was necessary prior to inoculation and enumeration (titers per liter). The distribution and abundance of phages infecting autochthonous bacterial hosts in a natural body of water, such as the Chesapeake Bay, illustrate the difficulty in interpreting data on the abundance of specific bacteriophages by culture assay (K. E. Wommack, R. T. Hill, J. Ravel, and R. R. Colwell, Abstr. 96th Gen. Meet. Am. Soc. Microbiol. 1996, abstr. N-23, p. 159, 1996). For example, 36 water samples collected at six stations during the year yielded only 10 samples that were positive for bacteriophages infecting one or more of the indicator strains. Of the 10 successful bacteriophage isolations from Chesapeake Bay water samples, only two of the titers exceeded the detection limit of 1 PFU. After taking into account the 10- to 100-fold concentration of virioplankton within the water samples, 7 PFU liter¹ was the final abundance estimate. However, direct microscopic examination revealed 100- to 1,000-fold more virus particles in each water sample.

Even though culture-based methods are not efficient in the enumeration of viruses, they remain indispensable for isolation and purification of virus-host systems. There are exceptions, however, most notably the methods used to study the distribution and abundance of phages of marine Synechococcus spp. Cyanophages that infect two Synechococcus strains isolated from Woods Hole harbor demonstrated stable annual cycles of abundance, ranging from winter lows of 10 ml¹ to late-summer highs of between 10³ to 10⁴ ml¹ (341). These abundances were recorded directly from analyses of seawater samples, without preconcentration, using a most-probable-number method and broth dilution cultures. Large numbers of cyanophage, often in excess of 10⁵ ml¹, have also been found in hundreds of water samples collected in the western Gulf of Mexico (96, 312, 313).

Obviously, culture-based methods have proven very useful in the study of cyanophage ecology. Cyanobacteria are a relatively well characterized group of aquatic bacteria. Synechococcus spp., used as host strains for titer determinations, were similar in genotype to members of the natural host community, a factor helpful in the detection of cyanophages in environmental samples. Waterbury and Valois (341) noted that the Synechococcus strains most successfully employed in titer determinations were those which had lost phage resistance, a result of a long time lapse (>2 years) since clonal isolation. The most-probable-number method was used to enumerate cyanophages (308), and broth culture may have enhanced the detection of these bacteriophages in natural waters. In contrast, the most-probable-number technique has been successful in enumerating bacteriophages infecting aquatic heterotrophic bacteria only in the case of Aeromonas spp. (85).

The majority of bacterioplankton has been presumed to consist of heterotrophic bacterial species (72, 128). However, very little is known about the community structure of heterotrophic bacteria, not even which species are numerically dominant. In retrospect, reliance on culturing methods can explain why nearly 35 years after Spencer (302) reported the isolation of a marine virus, i.e., Photobacterium phosphorium bacteriophage, the dogma persisted that viruses were rare in natural waters and therefore were without significance for their host populations. As we have known for over three decades, ever since the report of MacLeod (169), only a small proportion (1 to 2%) of naturally occurring heterotrophic aquatic bacteria can be grown in culture. Given that hundreds, if not thousands, of bacterial species are believed to comprise the bacterioplankton, any single bacterial species selected for estimation of phage populations will very probably not be a dominant species within the bacterioplankton host community. Therefore, phage titers, obtained with a few host strains, will underestimate virioplankton abundance.

Direct enumeration methods for viruses have facilitated the analysis of viral abundance in natural waters. Transmission electron microscopy is now used for direct enumeration of viruses, with one of the earliest transmission electron microscopy (TEM) observations of viruses in seawater samples having been made by Sieburth (292), who published a compendium on the diversity of marine microbial communities. In 1979, Torella and Morita (326) provided the first direct counts of viruses occurring within the >0.2-µm size fraction of plankton samples collected from Yaquina Bay, Oregon. At >10⁴ ml¹, the abundance of virus-like particles in filtrates of Yaquina Bay water was the highest recorded at the time. It was postulated that viral numbers were actually much larger, since many viruses pass through a 0.2-µm-pore-size filter. These findings provided the initial supporting evidence for the proposal of Weibe and Liston (354) that viral lysis and infection could both influence the growth and diversity of bacterioplankton and mediate genetic exchange. Despite these early, groundbreaking observations, active research on the ecological importance of viral infection languished for almost 20 years.

In 1988, two important studies showed that viral abundance in the range of 10⁶ ml¹ occurred in seawater. Using epifluorescence microscopy and a double-stranded DNA (dsDNA) binding fluorochrome, 4',6-diamidino-2-phenylindole (DAPI), Sieburth et al. (293) reported 5.8 × 10⁶ blue-fluorescing particles per ml in water samples collected during a monospecific bloom of a newly described chrysophyte alga, Aureococcus anophagefferens. Prior to enumerating free viruses in water samples collected during the bloom in Narragansett Bay, TEM observations of virus particles within Aureococcus anophagefferens cells had been made. In exploratory mesocosm experiments designed to investigate the potential of viral infection for bacterioplankton mortality, Proctor et al. (257) added virioplankton concentrates to water samples collected from the Eastern Caribbean. A significant impact of viral infection was apparent when, after a 24-h incubation period, the bacterioplankton abundance was 25 to 40% lower in mesocosms to which viruses had been added. TEM direct counts of virus-like particles in concentrates indicated a natural virioplankton abundance of between 10³ and 10⁶ ml¹.

Since direct counting can now be considered critical in studies of virioplankton ecology, comparison of the efficiency of methods used to count viruses is useful. Details of methods for direct counting of viruses are provided by Suttle (308, 309) and Bratbak and Heldal (35). The earliest and most commonly used method for viral direct counts (VDC) in water samples is TEM. From the results of studies by Proctor et al. (256, 257), Bergh et al. (19), and Sieburth (293), TEM has evolved into the benchmark for virus enumeration. Virus morphology, including capsid and tail size, can be determined by TEM.

The most commonly used TEM method is direct sedimentation of free virus particles from an unfiltered, glutaraldehyde-fixed water sample onto a fine-mesh, Formvar-coated copper grid. For most water samples, prefiltration prior to centrifugation is not necessary. However, there are technical as well as practical disadvantages of direct virus enumeration. In water samples containing large amounts of particulate matter, viruses are obscured on the grid and therefore cannot be counted. Because of this problem, viruses in water samples collected from the upper reaches of Chesapeake Bay could not be detected (368). Suttle (309) has addressed two additional problems in centrifuge pelleting of viruses onto TEM grids, namely, that viruses do not sediment in parallel paths and therefore are concentrated toward the edges of the bottom of the tube, and that very small viruses are not recovered with 100% efficiency. The result is an underestimation of virioplankton abundance. To minimize centrifugation bias, viruses are counted using replicate grids placed at the bottom of a centrifuge tube. Because of the high magnification required to identify virus-like particles on TEM grids, the practical detection limit of TEM direct counting is 10⁵ viruses ml¹, a detection limit high enough to prevent the application of TEM direct counting of samples collected from oligotrophic environments, where the number of viruses in the water is small (138). Finally, there is loss of precision because of uneven staining and nonhomogenous distribution of viruses on the grids. TEM counts of viruses in natural water samples yield an average coefficient of variation of 20 to 25%, versus 7 to 11% for epifluorescence microscopy (122, 349).

To enumerate viruses in samples of oligotrophic water without having to centrifuge the samples, virioplankton can be concentrated by ultrafiltration. In fact, ultrafiltration is essential in virioplankton ecology. Methods for ultrafiltration and concentration of naturally occurring viruses include vacuum filtration (367), centrifugal filtration (97), pressure filtration through either a hollow-fiber filter (254, 256), or a spiral-wound membrane filter (52, 316), and vortex flow filtration, a variant of pressure filtration (139, 240). Viruses in virioplankton concentrates are either pelleted during centrifugation or dried onto a TEM grid and then stained. Ultrafiltration, while improving the detection and enumeration of virioplankton and perhaps even eliminating centrifugation, introduces a selective bias. As a step in obtaining direct counts of viruses, it can affect recovery efficiencies, e.g., 9 to 117% ( = 42 ± 42) for vortex flow filtration (138, 240) and 68 to 171% ( = 114 ± 30) (mean ± standard error) for vacuum filtration (367), indicating low but variably reduced counts. The strongest evidence of ultrafiltration bias was obtained by analysis of covariance, comparing direct counts of viruses with and without ultrafiltration. Overall, viral abundances estimated using unprocessed samples were nearly three times greater than those estimated using concentrated samples (171). Methodological limitations aside, ultrafiltration has made possible the examination of virioplankton in both oligotrophic water samples and mesocosms in which virus-host population concentrations have been altered. In general, ultrafiltration provides conservative virus counts.

The practical limitation of TEM direct counting is the equipment required, i.e., a transmission electron microscope and ultracentrifuge. Thus, it is not suitable for most field studies and is less likely to be available to investigators at small research institutions. Also, sample preparation and analysis are tediously slow. These limitations can be bypassed by using epifluorescence light microscopy (ELM). In essence, the latter is a variation of direct counting of bacteria. Nucleic acid within the viral capsid is stained with a nucleic acid binding fluorochrome (usually dsDNA). Stained viruses are captured on a small-pore-size filter (0.02 µm) and visualized at high magnification (×1,000) by light emission of the bound fluorochrome stain. Successful enumeration of viruses generally requires intensification of the fluorescent signal by means of a charge-coupled device camera (97) or photographic processing designed to increase film speed (116). Several different dsDNA binding fluorochromes, e.g., DAPI, Yo-Pro, and SYBR, have been used as the staining agent. An obvious caveat for those who use ELM to count viruses is that the overlap in size between small bacterioplankton cells and large virus-like particles, in some environments, can be a source of error in direct counts of viruses (299).

With improvements in the specificity and fluorescence yield of dsDNA binding fluorescent stains, ELM methods for direct counting of virioplankton now approach a precision level similar to that of TEM. Initial trials, using DAPI (Table 1), found relatively close agreement between TEM and ELM counts of viruses in Japanese coastal water samples (TEM/ELM ratio  0.8) (115, 116), in phage lysates of laboratory cultures (TEM/ELM ratio  1) (254), and in virioplankton concentrates from Santa Monica Bay (TEM/ELM ratio  1.6 ± 0.37) (97). In only one study, using water samples from the Gulf of Mexico and Tampa Bay, did TEM counts greatly exceed ELM (DAPI) counts (TEM/ELM ratio  10 to 18) (240). Discrepancies among these studies probably can be ascribed to methodological differences, especially for the Gulf of Mexico and Tampa Bay samples, where image intensification was not utilized.

More recently, two other nucleic acid binding fluorochrome stains, Yo-Pro-1 and SYBR Green I, have been used for virioplankton enumeration (144, 173, 214). Specific advantages of these stains are low background staining and stability and brightness of fluorescence greater than that of DAPI. A notable disadvantage of Yo-Pro-1 is the 2-day incubation needed for adequate staining of virioplankton samples trapped on an aluminum oxide filter. Subsequently, it was reported that the cumbersome 48-h incubation time could be reduced to 4 min through microwave pretreatment of virioplankton samples (376). In several trials, Hennes and Suttle (122) found that ELM (Yo-Pro) virus counts exceeded those of TEM by a factor of ca. 3 ± 1.6. Similarly, Weinbauer and Suttle (349) observed that estimates of virioplankton abundance obtained by TEM averaged ca. 66% of those obtained with ELM (Yo-Pro). For the same Gulf of Mexico water samples, ELM virus counts obtained using DAPI staining averaged 86% of the Yo-Pro counts.

Two recent reports indicate that SYBR Green I may emerge as the best solution for epifluorescence counting of virioplankton (173, 214). Unlike Yo-Pro, SYBR Green I stains only viruses and cells in seawater samples with a short (<15-min) incubation and is not affected by the presence of aldehyde fixatives (214). As with Yo-Pro, SYBR Green I virus counts yielded a precision similar to that of TEM counts and generally exceeded TEM direct counts. In a most exciting methodological advancement, SYBR Green I staining has been combined with flow cytometry for rapid enumeration of virioplankton in seawater samples (173). Flow cytometric analysis of virioplankton populations in water samples collected from different depths in the Mediterranean Sea revealed two distinct virus populations, based on size. If the majority of the bright, Yo-Pro-1- or SYBR Green I-stained particles are viruses, most virus counts obtained through TEM (Table 1) are very conservative and probably underestimate the actual population size.

It is likely that virioplankton, besides being most abundant, is the most diverse component of plankton. Hundreds of viruses infecting a wide range of phyto- and bacterioplankton species have been isolated and described. However, as with aquatic bacteria, these represent only a small portion of virioplankton diversity. For example, recently it was discovered that in some marine environments, archaebacteria make up a significant proportion of the bacterioplankton (69, 70). The archaea were previously believed to be restricted to extreme environments characterized by high salinity, high temperature, or anaerobiosis (224). Only a few viruses infecting archaebacteria have been described (380) because of the difficulty of culturing the hosts and the slow growth of archaeal viruses. Archaea make up to 34% of bacterioplankton abundance in Antarctic coastal waters (70) and 40 to 60% of total bacterial abundance in temperate ocean midwaters (200 to 600 m) (94); therefore, it is probable that archaeal viruses constitute a significant proportion of the virioplankton in these environments.

At present, direct TEM examination of aquatic viruses is the most common method of recording virioplankton diversity. While morphological data give only a limited view of virioplankton diversity, these data have been cited as evidence that bacteriophages comprise the majority of viruses within the virioplankton (172, 368). Other observations, such as the lack of significant correlation between algal biomass (chlorophyll a concentration) and VDC (Table 1), the ability of changes in bacterial abundance to predict changes in viral abundance (56, 115), the greater abundance of bacteria over that of other planktonic hosts (28, 56), and the predominance of viruses within the virioplankton with bacteriophage-sized genomes (20 to 60 kb), have all been cited as evidence to support this claim (116, 372). Further study may fully substantiate the prevalence of bacteriophages within the virioplankton. The morphology of marine bacteriophages has been reviewed extensively by Proctor (257), Børsheim (29), and Frank and Moebus (86).

Capsid size provides an index of morphological diversity. Classification according to capsid size has been accomplished, based on data obtained by TEM. In fact, viruses in environmental samples have been classified according to morphotype by some investigators (353). The most complete of these studies was a survey of bacteriophage diversity in Lake Plußsee, Germany, by Demuth et al. (71), who reported 39 morphologically distinct phages, which were classified into three principal morphotypes: B1 (Siphoviridae), A1 (Myoviridae), and C1 (Podoviridae). These morphotypes made up 50, 18, and 19% of the total diversity, respectively.

Other studies have reported only on the incidence of tailed bacteriophages. Hara et al. (116) and Wommack et al. (368) showed quantitatively that nontailed or short-tailed viruses comprised the majority of virioplankton in Japanese coastal and Chesapeake Bay water samples, respectively. Other investigators have reported (qualitatively) that either long-tailed (contractile and noncontractile) viruses (56, 256, 326) or non- or short-tailed viruses (19, 38) were dominant in water samples. The accuracy of reports on tail morphology, however, has been questioned, since preparation of water samples for viral direct counting may cause separation of phage capsids and tails (253). Since 96% of all known bacteriophages (ca. 4,600) are tailed (1), it is not surprising that careful preparation and exhaustive documentation of virioplankton diversity in aquatic environments (such as that done by Demuth et al. [71]) show that the majority of virioplankters are tailed.

Measuring virioplankton diversity by capsid diameter is appropriate, since this feature varies widely among the bacteriophages described to date (20 to 200 nm) and is, in general, a consistent feature for bacteriophages (3). Data on the frequency distribution of capsid size within virioplankton populations have been reported for a variety of aquatic environments (Table 1), with the dominant virioplankton capsid diameter being in the range of 30 to 70 nm (19, 30, 38, 56, 121, 171, 172, 320, 342, 347, 368). The proportion of virioplankton observed to fall into the 30- to 60-nm size class was ca. 65% or greater. Two investigators have reported the predominance of 30- to 60-nm viruses within bacterioplankton cells (121, 347), suggesting that the free bacteriophages most frequently observed and characterized are indeed produced in situ. The 30- to 60-nm capsid size of aquatic virioplankton is slightly smaller than the 60- to 80-nm range observed for purified marine (29, 86) and other (3) bacteriophages. It has been suggested that the somewhat smaller size of free aquatic bacteriophages indicates that marine phage culture collections do not contain the most abundant bacteriophages that exist in aquatic environments (29). Therefore, conclusions about natural bacteriophage diversity that are based on knowledge gained from experiments with purified bacteriophages must be drawn with caution. For example, Bratbak et al. (34) observed large numbers (10⁴ ml¹) of extraordinarily large phage-like particles, with a capsid diameter of 340 to 400 nm and noncontractile tail length of 2.2 to 2.8 µm, in Norwegian and Danish coastal water samples. Abundant large virus particles that overlap the size of small bacteria have also been found in water samples from a eutrophic freshwater reservoir (298), Antarctic lakes (144), European alpine lakes (249), and the food vacuoles of phaeodarian radiolarians (111). Pleiomorphic, spindle- and lemon-shaped viruses have been found in hypersaline environments dominated by halophilic archaea (113, 227).

Changes in the frequency distribution of viral capsid size over time and space suggest that the composition of virioplankton populations and, by inference, cooccurring host populations can vary. Evidence for a correlation between the capsid size of the virioplankton and the composition of host populations comes from the observation of Weinbauer and Peduzzi (347), who showed that bacteria within different morphological groups carry viruses of a particular size class. Analysis of Adriatic Sea water samples revealed that the 30- to <60-nm capsid size class comprised 75 and 100% of intracellular viruses in rods and spirilla, respectively. Cocci, however, more often contained larger viruses (60 to <110 nm) than the smaller 30- to <60-nm viruses (65 and 35%, respectively). Within the bacterioplankton, 84% of the population was rod shaped, supporting the results of earlier work showing a predominance of 30- to <60-nm capsids within virioplankton from Northern Adriatic Sea water samples (342).

Several observations of temporal and spatial changes in the frequency distribution of viral capsid size have been recorded for virioplankton populations. Cochlan et al. (56) found that viruses >60 nm in capsid diameter decreased greatly in abundance, from nearly 50% of the population to almost none, in an onshore-to-offshore transect in the Southern California Bight. The authors observed that differences in virioplankton abundance from water samples collected at different depths or in different environments (Southern California Bight and the Gulf of Bothnia, Sweden) could usually be attributed to the proportion of viruses in the <60-nm size class. A similar observation of the dynamic nature of the <60-nm capsid size class was made by Børsheim and colleagues (30) during a 20-day incubation of a Norwegian coastal seawater sample. In the first 5 days of incubation, the abundance of the <60-nm class increased at a rate of 41% per day. Two studies have documented changes in both virioplankton abundance and composition during Northern high-latitude spring diatom blooms. Bratbak et al. (38) observed two peaks in virioplankton abundance over a 1-month sampling period, during which virioplankton in the <60-nm size class were nearly always dominant and demonstrated the most erratic changes in abundance. Maranger et al. (172) noted a decrease in the abundance of the smallest size class (viruses of <50 nm) and an increase in the 50- to 70-nm size class in samples of Arctic sea ice during a 1-month bloom.

Not every study documenting the capsid diameter of virioplankton has reported changes in the distribution of capsid size with changes in time or location of water sample collection. Mathias et al. (176) noted no change in the frequency of four groups of capsid diameters among the virioplankton in river water samples collected over a 2-year period. It is interesting that viruses larger than 60 nm were most abundant in these water samples (mostly the 60- to <90-nm size class [ca. 40%]). The preponderance of larger viruses in the virioplankton of this riverine environment may be a reflection of differences in host community structure between marine and freshwater environments. In the hypersaline environment of solar salterns, the virioplankton members tend towards smaller capsid diameters, with the dominant size class between 20 and 50 nm (113). In this extreme environment, no correlations were observed between changes in salinity and frequency of capsid diameter; however, the abundance of a pleiomorphic, lemon-shaped virus (believed to be a virus of halophilic archaea) was strongly correlated with increasing salinity (113). Differences in viral size class distribution was noted for water samples collected from 22 Canadian lakes; however, no statistically significant trend connecting these changes to the trophic state of the lacustrine environment was detected (171). Similarly, the frequency distribution of capsid diameters did not change significantly with season or trophic condition for virioplankton populations in water samples from the Northern Adriatic Sea (342).

In the few years since the presence of large numbers of free virus particles in natural waters was reported, microbial ecologists have used a variety of direct-counting techniques to enumerate viruses in the aquatic environment. As shown in Table 1, the abundance of viruses can range from <10⁴ ml¹ to >10⁸ ml¹. This extreme variability in numbers is distinctive for this component of the plankton. In similar environments, the number of bacteria ranges between ca. 10⁴ and 10⁷ ml¹, with ca. 10⁶ ml¹ reported for coastal marine waters. The data given in Table 1 show that the number of viruses is greater in productive and nutrient-rich environments. The wide variation in numbers suggests that viruses are, indeed, an active component of aquatic microbial communities.

Seasonal variation in abundance has been noted since the earliest reports of viruses in seawater by Bergh et al. (19). In Norwegian coastal waters, the concentration of virus particles during the winter fell below 10⁴ ml¹ from ca. 5 × 10⁶ ml¹ in spring through autumn. Counts made at other coastal ocean and estuarine locations have shown similar seasonal trends. In both Tampa Bay and the northern Adriatic Sea, viral abundances changed by an order of magnitude from winter lows of 10⁶ ml¹ or less to late-summer highs of >10⁷ ml¹ (58, 136, 343). Significant seasonal changes in viral abundance were observed in Chesapeake Bay (368). In the only study done to examine the seasonal dynamics of a freshwater virioplankton population, Mathias et al. (176) recorded viral abundances ranging from 12 × 10⁶ to 61 × 10⁶ ml¹ in a backwater system of the Danube River. Bacterial abundances showed similar seasonal variability in these environments but changed only by a factor of 5. An interesting coincidence between these studies is that the greatest viral abundance occurred in late autumn. It is possible that these annual peaks in viral abundance are a result of autumn phytoplankton blooms. Spring phytoplankton blooms also are correlated with increased numbers of viruses; however, the magnitude of the autumn peaks is larger, possibly because of larger initial (summer) virioplankton and bacterioplankton populations at the beginning of the autumn bloom.

The volatile nature of virioplankton abundance is most apparent in shorter-term temporal studies, especially those conducted on or around annual phytoplankton blooms. Initial evidence that viruses were likely to be active within the plankton came from a study examining changes in numbers of free virus particles during a spring diatom bloom in western Norway. Over the course of the March 1989 bloom, employing 2-day sampling intervals, Bratbak et al. (38) documented a rise in numbers of viruses from a prebloom low of 5 × 10⁵ virus-like particles ml¹ to a maximum concentration of 1.3 × 10⁷ virus-like particles ml¹. Dramatic peaks in free-virus counts followed peaks in the abundance of diatoms and other photoautotrophs, suggesting a close coupling of viral infection and production to growth of the host population. In a 4-month study of Lake Constance, a large mesotrophic lake, Hennes and Simon (121) documented changes in virioplankton abundance, frequency of infected bacterioplankton, and bacteriophage production rate. Their observations, which commenced just prior to the spring phytoplankton bloom, showed that transient increases in bacterial abundance were closely followed by peaks in the frequency of occurrence of infected bacteria and in the abundance of free bacteriophages. Brief peaks in virioplankton abundance suggested in situ phage production rates of between 0.5 × 10⁶ and 2.5 × 10⁶ phage ml¹ day¹. Finally, dramatic changes in virioplankton abundance were observed in Arctic sea ice over the course of the spring phytoplankton bloom. In observations of virioplankton in the lower 4 cm of sea ice, Maranger et al. (172) reported the largest numbers of free virus ever recorded in a marine environment. During the 1-month bloom, viral abundance in sea ice increased by a factor of 100. Surprisingly, viruses were 10 to 100 times more abundant in sea ice than in the underlying water, suggesting that processes controlling virus populations are dramatically different in the two environments.

Short-term changes in virioplankton abundance have been investigated both in large-volume (60-liter) mesocosms and in situ. In each case, the number of free viruses in water samples changed significantly over a diel cycle. A mesocosom experiment conducted by Jiang and Paul (136) did not reveal a diel rhythm in changes in virus numbers. During the experiment, a significant increase in VDC was preceded by increases in bacterial abundance and chlorophyll a concentration, thereby providing circumstantial evidence for close coupling of host community growth and viral production (136). A diel cycle of viral abundance that was significantly correlated with sunlight levels and bacterial production was detected in 60-liter seawater enclosures (120). In the enclosures, a direct link between host and viral abundance was not observed, since the bacterial direct count (BDC) did not correlate with VDC. However, a correlation of the VDC with the bacterial production rate in the mesocosms supports the assumption that a faster-growing host community is more effective in producing free viruses. Correlation of VDC with global radiation levels led Heldal and Bratbak (120) to suggest that in the microcosms, sunlight stimulated viral production by induction of lysogenic bacteria. In one instance, a significant fluctuation in virioplankton abundance was noted during intervals as short as 10 min. Bratbak et al. (40) recorded two- to fourfold changes in VDC over periods of 10 to 20 min in seawater mesocosms. These short-term changes indicated extremely high viral production (6 to 13 h¹) and loss (5 to 11 h¹) rates. These radical changes in VDC were interpreted as an indication of synchronous lysis of bacterioplankton hosts.

A limitation of mesocosm studies is the experimental error arising from enclosure of the planktonic communities. Many investigators acknowledge that the "bottle effect" can influence conclusions drawn from mesocosm data for natural populations of microorganisms (383). However, observations of virioplankton dynamics in seawater incubations do not indicate effects related to confinement. Rapid changes in virioplankton abundance recorded by Bratbak et al. (40) were not affected by the volume of seawater in mesocosms (ranging from 30 ml to 20 liters), leading the authors to dismiss the bottle effect as an explanation for high short-term virioplankton production rates. Weinbauer et al. (343) monitored diel in situ variability in virioplankton density for 42 h and found changes in VDC and other parameters. Peaks in BDC and chlorophyll a concentration preceded significant changes in VDC. These findings, along with those of Jiang and Paul (136) and Hennes and Simon (121), indicate that short-term changes in virioplankton abundance, observed in large-volume mesocosoms, reflect in situ processes.

Physiochemical changes with depth can have a significant impact on planktonic microorganisms. Within the oceanic euphotic zone, specific distributional patterns of photoautotrophs occur that are related to light intensity and wavelength (225, 226). Water temperature or salinity gradients (clines) can result in water column stratification into separate physiochemical environments. For instance, in a salt wedge estuary such as the Chesapeake Bay, the water column is stratified by a strong halocline, separating denser seawater from seaward-flowing freshwater. During calm summer weather, the more saline bottom waters of the Chesapeake Bay become anoxic, which, in turn, affects biological processes significantly (218, 330).

In open-ocean waters, virioplankton abundance declines rapidly below the euphotic zone (200 m) to relatively constant, low abundances of <10⁶ viruses ml¹ (25, 28, 115, 240, 304). In one instance, deep-water viral abundance, at a station in the Southern California Bight, was between 1.1 × 10⁶ and 2.5 × 10⁶ ml¹ and showed an increase in near-bottom (900 m) water samples (56). Within the upper 200 m, transient subsurface maxima in viral abundance can occur, usually at ca. 15 m (25), 50 m (28, 56, 115) and 75 m (25) to 150 m (28, 56, 115, 304) in the water column. Hara et al. (115) observed subsurface peaks in virioplankton abundance in the north Pacific, above and below the depth of the subsurface chlorophyll maxima. The discontinuity of viral abundance around the subsurface chlorophyll maxima suggests that there are changes in the processes responsible for virioplankton production or loss between 50 and 150 m deep in the open ocean. Cells transported out of the high-productivity zone may be exposed to nutrient limitation or changes in light intensity and therefore may be more susceptible to viral infection or to curing of the lysogenic state, resulting in higher rates of viral production.

Unlike deep-ocean waters, where viral counts at depth are 2 to 10 times lower than surface counts, near-coastal and estuarine water often does not show significantly fewer viruses with depth (56, 244, 343, 368). Consistently large numbers of viruses (usually 10⁷ viruses ml¹) throughout the water column are observed in near-shore environments. Nevertheless, in a few instances, depth-related variability in VDC has been observed in productive waters. During periods of water column stratification in the northern Adriatic Sea, virioplankton abundance is significantly greater at the thermocline (343). In a permanently stratified Norwegian lake, viruses and bacteria were two- to threefold more abundant at the chemocline (the boundary layer separating anoxic, sufidic bottom water from oxic surface water) than at the surface (329). This observation is not surprising, since bacterioplankton, phytoplankton, and nutrients are known to concentrate within the boundary layer of a stratified water column (189). Similarly, the surface microlayer of natural waters is known to be a zone of high bacterial productivity. In the only report of virus abundance in the surface microlayer, Tapper and Hicks (320) found that the highest abundances of virioplankton in Lake Superior water samples were from the upper 20 µm of the water column. With water column mixing, however, the subsurface thermocline peak in the virus-to-bacterium ratio dissipates and the virioplankton concentration is generally uniform throughout the water column (343, 368). Stratification and deep-water anoxia in Chesapeake Bay were observed to have a significant impact only on bacterioplankton abundance, whereas virioplankton abundance at depth was similar to that at the surface (368). However, the viral abundance at the boundary layer was not measured. Altogether, temporal and depth-related variability in virioplankton abundance provides evidence that viruses play an active and important role in aquatic microbial communities.

Chlorophyll a concentration and bacterial abundance. The unifying message of the data presented in Table 1 is that the production and distribution of viruses in aquatic environments is, not surprisingly, determined by factors which affect the productivity and density of host populations, especially the bacterioplankton. In most instances, where changes in virioplankton abundance and chlorophyll a concentration were recorded, no significant correlation was observed between these parameters. Conversely, in a majority of studies which have examined changes in BDC and VDC, significant correlation between bacterial and viral abundance have been observed (28, 56, 115, 121, 136, 144, 243, 304, 329, 342, 343, 348). Moreover, in nearly every environment where chlorophyll a concentration significantly predicted virioplankton density, bacterial abundance did so as well (28, 136, 343).

Bacterial production. It is possible that the confusion in determining factors that are significantly related to virioplankton distribution will be cleared up by examining the correlation of bacterial activity (production) with viral abundance. On the few occasions where viral abundance and bacterioplankton production have been measured simultaneously, significant correlations have been observed (Table 1) (120, 171). Other supporting evidence that bacterial activity can be correlated with virioplankton abundance is derived from data obtained from Arctic sea ice, where the region of maximal bacterial production corresponds to the region of highest viral count (172). A more direct indication that increased viral production coincides with the level of bacterial secondary production was noted by Steward et al. (304). A significant correlation (r² = 0.64) was observed between the frequency of visibly infected cells (FVIC) (measured by TEM) and bacterial production rates (304). While it would appear obvious that a link between viral abundance and rates of bacterial production should exist, a clear demonstration of such a link provides strong evidence that maintenance of abundant virioplankton populations is dependent on an active bacterioplankton host community.

The dissolved-DNA dilemma. In addition to chlorophyll a levels, BDC, and bacterial production measurements, the connection between virioplankton abundance and dissolved-DNA (D-DNA) concentration has been investigated. Interest in the abundance and distribution of DNA in aquatic environments has been fueled both by research on the biological cycling of carbon through aquatic microbial communities and concerns about the consequences of the release of genetically engineered microorganisms into the environment. In the first instance, D-DNA (the DNA fraction which passes through a 0.2-µm-pore-size filter) is a readily measurable portion of dissolved organic matter (DOM) which is useful in studies of DOM dynamics. Because of its ubiquity in living cells and its specialized role in cell function and reproduction, DNA also serves as a useful tracer molecule for analyzing planktonic rate processes (142). An excellent model of the distribution of total DNA (particulate and dissolved) in the oceanic environment has been provided by Jiang and Paul (138). Secondly, the possibility has been offered that D-DNA serves as a reservoir through which exotic or engineered gene sequences could be introduced via transformation into autochthonous, aquatic bacteria (68, 238).

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In addition to correlations between VDC and environmental parameters, the virus-to-bacterium ratio (VBR) has been used to study the relationship between virioplankton and bacterioplankton populations. From VBR data presented in Table 1, it is easy to appreciate that even in oligotrophic environments, viral abundance exceeds bacterial abundance. With the sole exception of Lake Superior waters, where VBR values were consistently low (i.e., <1) (320), generally the ratio of virus to bacterial abundance falls between 3 and 10. VBR values are higher for more nutrient-rich, productive environments. This simple observation suggests that bacterioplankton host populations produce greater numbers of viruses under environmental conditions favoring fast growth and high productivity. These increases in virioplankton production under nutrient-replete conditions are also due, in part, to higher infection rates and larger burst sizes, two biological parameters which can strongly influence VBR. VBR can be useful in constructing theories on the effect of viral infection on aquatic bacterial host communities; however, it is important to also consider that this ratio can be influenced by a multitude of factors which control the production and loss of viruses and bacteria.

Studies of changes in VBR have revealed several associations between VBR and bacterial density. Results of field studies off the coast of Japan (116) and in a Norwegian meromictic lake (329) revealed that while BDC and VDC change dramatically with depth and over short temporal scales, VBR values remained consistent, suggesting a tight coupling between bacterial and viral concentrations and relatively constant levels of virus production and loss. An unchanging phage-to-bacterium ratio was also noted by Ogunseitan et al. (220) in freshwater mesocosm studies of Pseudomonas aeruginosa and bacteriophages UT1 and M1.

A positive relationship between VBR and bacterial numbers was noted in both oligotrophic water samples collected in the North Pacific (115) and eutrophic water samples collected from the northern Adriatic Sea (343). Hara et al. (115) suggested that the positive relationship between these parameters in Pacific Ocean water samples arises from a direct dependence of virus production on bacterial host cell density. They also suggested that, based on the observation that higher VBR values coincided with larger numbers of small bacteria, increased numbers of viruses may act as a positive selective pressure for reduced cell volume. This suggestion is also supported by the colloidal aggregation theory, which predicts that a smaller bacterium would encounter fewer phages through Brownian motion (115).

While these studies offer intriguing theories, they represent special cases of viral and bacterial community interaction. The most commonly observed relationship between VBR and bacterial abundance is either negative or inverse. Field studies of Tampa Bay (136), Arctic sea ice (172), Canadian lakes (171), and seawater mesocosms (328) have all provided results showing that the VBR decreases as bacterial abundance increases. In our analysis of Chesapeake Bay, we noted that the VBR was inversely related to bacterial abundance (368). The highest VBR values were recorded at times of relatively low bacterial abundance and vice versa, suggesting seasonality in virioplankton production in the nutrient-rich ecosystem of Chesapeake Bay. Seasons where a high VBR occurs would show more virioplankton production and hence more bacterioplankton lysis. Maranger et al. (172) reported an extremely high VBR of 72 in sea ice at the onset of the spring phytoplankton bloom, which declined precipitously as bacterial numbers increased. They speculated that as the bloom proceeded, the pressure of viral infection selected for phage-resistant mutant bacteria among the bacterioplankton. Toward the end of the bloom, resistant bacteria were dominant among the sea ice bacterioplankton, resulting in reduced active production of phage. The dominance of resistant bacteria could also explain the lower bacterial production rates recorded at the end of the bloom. Phage resistance often results in lower physiological efficiency (160).

Finally, Bratbak and Heldal (36) and Tuomi et al. (328), in explaining the negative or inverse relationships they observed between VBR and bacterial abundance, formulated the intriguing hypothesis that the VBR may be an indicator of host community diversity. In a bacterioplankton community dominated by only a few bacterial species, specific adsorption of viruses to their cooccurring host would be favored. Once adsorbed to their specific receptor, free viruses are effectively lost from the virioplankton. In environments with high host diversity, virioplankton would presumably spend more time as free viruses, since specific adsorption would be slower. This argument hinges on two plausible assumptions: (i) that specific adsorption is an important factor in viral loss, and (ii) that bacterial numbers increase as the overall community diversity decreases. The latter assumption can easily be accepted for mono- or polyspecific blooms, when populations can dramatically increase in number, with simultaneous loss in overall species diversity.

IMPACT OF VIRUSES AND VIRAL INFECTION ON THE AQUATIC FOOD WEB

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Significant Biological Factors for Modeling In Situ Phage-Host Interaction

In situ burst size. Burst size, i.e., the number of virus particles released upon host cell lysis, is an essential component in calculations that relate estimates of in situ viral production to the level of virus-mediated mortality of bacterioplankton. Intuitively, there is an inverse relationship of burst size and the level of host mortality due to viral infection. The greater the burst size, the smaller the number of host cells lysed to support a given level of virus production. Noting the dramatic effect of burst size estimates on modeling the impact of viral infection on host populations, many authors have chosen to use a range of burst sizes for these calculations (see Tables 2 through 6). However, it is likely that improvements in the precision of estimates of virus-mediated mortality can most easily be achieved through more accurate estimates of burst size under in situ conditions. To this end, several studies have used burst size estimates, based on in situ observations of bacterio- and phytoplankton, to calculate virus-mediated mortality. Burst size estimates are summarized in Table 2.

Viral inactivation and particle destruction. Since the earliest studies of bacteriophage physiology (79), researchers have routinely reported data on the stability of purified viral isolates. Initially, data on viral inactivation were of practical use only in research; however, public health concerns about the safety of recreational, drinking, and shellfish-producing waters soon added significance to investigations on the survival of viruses in aquatic environments. Thus, most studies on the survival and fate of viruses in natural waters has focused directly on enteroviruses or on coliphages used as indicators of enteroviral pollution. The latest stimulus for investigating virus decay and destruction in natural waters has come from interest in the impact of viral infection on bacterial productivity. If, as enumeration studies seem to indicate, virioplankton abundance is relatively stable over seasonal scales, rates of viral production and loss should be equal. Therefore, estimations of viral loss and inactivation in aquatic environments should indicate the virus production rate needed for steady-state conditions. In turn, estimates of viral loss rates can lead to estimates of the level of virus-mediated mortality of bacterioplankton hosts. For excellent, comprehensive reviews of the early literature on viral inactivation, see references 5 and 141.