Abundance and Distribution of Synechococcus spp. and Cyanophages in the Chesapeake Bay
ABSTRACT
Despite the increasing knowledge of Synechococcus spp. and their co-occurring cyanophages in oceanic and coastal water, little is known about their abundance, distribution, and interactions in the Chesapeake Bay estuarine ecosystem. A 5-year interannual survey shows that Synechococcus spp. and their phages are persistent and abundant members of Chesapeake Bay microbial communities. Synechococcus blooms (106 cells ml−1) were often observed in summer throughout the Bay, contributing 20 to 40% of total phytoplankton chlorophyll a. The distribution of phycoerythrin-containing (PE-rich) Synechococcus cells appeared to mostly correlate with the salinity gradient, with higher abundances at higher salinities. Cyanophages infectious to Synechococcus were also abundant (up to 6 × 105 viruses ml−1 by the most probable number assay) during summer months in the Bay. The covariation in abundance of Synechococcus spp. and cyanophages was evident, although the latitude of observed positive correlation varied in different years, mirroring the changing environmental conditions and therefore the host-virus interactions. The impacts of cyanophages on host Synechococcus populations also varied spatially and temporally. Higher phage-related Synechococcus mortality was observed in drought years. Virus-mediated host mortality and subsequent liberation of dissolved organic matter (DOM) may substantially influence oceanic biogeochemical processing through the microbial loop as well as the microbial carbon pump. These observations emphasize the influence of environmental gradients on natural Synechococcus spp. and their phage population dynamics in the estuarine ecosystem.
INTRODUCTION
Unicellular cyanobacteria of the genus Synechococcus are among the most abundant (up to 105 ml−1) and ubiquitous members of the picophytoplankton in the open ocean (15, 44, 46) and are responsible for ca. one-fourth of primary production in the world's ocean (17, 18, 33, 46).
Synechococcus spp. are also abundant in various estuarine ecosystems, including Chesapeake Bay (2, 36), Southampton Estuary (12, 13), Bay of Blanes (3), Florida Bay (34), San Francisco Bay (28), Pensacola Bay (27), and Changjiang Estuary (32). Synechococcus cell density typically ranges from 102 to 105 cells ml−1 in temperate estuaries but often exceeds 106 cells ml−1 in subtropical regions (27). In temperate estuaries, the composition of phycoerythrin-rich (PE-type) versus phycocyanin-rich (PC-type) Synechococcus spp. appeared to vary with the salinity gradient (12) and with temperature (2), i.e., a higher PE-to-PC ratio was often seen in higher-salinity water. The 6- to 7-month water residence time in the Chesapeake Bay is at least 1 to 2 times longer than those in other estuaries (29), allowing for the development of indigenous bacterial species in the Bay (16).
Estuarine ecosystems are complex and can be unique from one to another in terms of environmental conditions. Chesapeake Bay is subject to various climatic forces that influence the growth and distribution of phytoplankton (20). Similar to other temperate estuaries, the annual temperature varies from 0 to 30°C in the Chesapeake Bay, yet the annual cycle of freshwater discharge typically demonstrates a spring maximum and a summer minimum in the Bay. Over half of the annual nutrient input to Chesapeake Bay occurs during spring runoff (March through May). Consequently, the excess nutrient input often triggers phytoplankton (mainly diatom and dinoflagellate) blooms in the spring (20). Earlier studies reported that picophytoplankton (dominated by Synechococcus) could contribute 10 to 14% of Chesapeake Bay primary production during early summer (36). During late summer, Synechococcus can reach nearly 106 cells ml−1 and account for 56% of primary production in the southern part of the Bay (1). Picoplankton productivity often exceeds heterotrophic bacterial productivity during summer months in the Bay (21). The relative contributions of PE-type and PC-type Synechococcus spp. vary seasonally in the lower Chesapeake Bay (2). Previous studies of Chesapeake Bay Synechococcus spp. focused mainly on specific regions of the Bay and were not very systematic. A large-scale and long-term survey of the spatial and temporal dynamics of Synechococcus in the Chesapeake Bay has not been undertaken. Thus, interannual variations in Synechococcus abundance and the mechanisms influencing such variations still remain unclear.
Grazing and viral lysis are the two main factors responsible for microbial mortality in aquatic environments (10, 30, 47). Recent studies showed that Synechococcus appears to be a poor food source for nanoflagellates (7). Only 0.2 to 1.0% of natural Synechococcus populations are consumed by grazing (9). In contrast, the lysis of Synechococcus organisms by cyanophages (viral pathogens of cyanobacteria) can have a significant impact on the mortality of Synechococcus (22, 39). It was estimated that viral lysis could cause up to a 5 to 14% loss of Synechococcus cells in the open ocean on a daily basis (40). In coastal water, cyanophage titers covary with Synechococcus abundance on temporal and spatial scales (23, 41, 45). The abundance of infectious cyanophages was found to increase along with salinity in the Savannah River estuary (19).
Early studies suggested that virioplankton could be an important factor affecting the biomass and population structure of bacterioplankton in the Chesapeake Bay (48, 49). While the relationship between total viral and bacterial counts in the Chesapeake Bay has been studied (48, 50), only a few studies have been conducted to understand the interaction between specific virus and host systems in the Bay (42). Our recent explorations have uncovered many novel and diverse Synechococcus spp. in the Chesapeake Bay (6), whereas numerous lytic cyanophages belonging to the three double-stranded DNA (dsDNA) virus families (i.e., Myo-, Podo-, and Siphoviridae) have been isolated using endogenous Synechococcus strains from the Bay (43), implying that cyanophages can be active and important pathogens affecting host populations. Synechococcus spp. and their phages are an ideal system for studying host-virus dynamics at the population level, because (i) they are abundant and dynamic in the marine environment, (ii) total Synechococcus organisms can be counted based on their autofluorescence, and (iii) cyanophages can be titrated by use of representative host cultures.
To better understand the ecological impacts of cyanophages on picocyanobacteria in the estuarine ecosystem, we comonitored the spatial and temporal distributions of Synechococcus spp. and their co-occurring cyanophages in the Chesapeake Bay for five consecutive years (September 2002 to February 2007). We attempted to find out how and to what degree viral lysis would affect Synechococcus populations under different environmental conditions and, ultimately, how this would affect primary production in the Chesapeake Bay ecosystem.
MATERIALS AND METHODS
Sample collection.
Water samples were collected from nine stations along the midstem of Chesapeake Bay by researchers on board the R/V Cape Henlopen and R/V Hugh R. Sharp during the research cruises for the MOVE (Microbial Observatory of Virioplankton Ecology) project, from September 2002 to February 2007 (details of these cruises can be found at http://www.virusecology.org/MOVE/Home.html).
The locations of the nine stations are shown in Fig. S1 in the supplemental material. Their coordinates are as follows: station 908, 39°08′N, 76°20′W; station 858, 38°58′N, 76°23′W; station 845, 38°45′N, 76°26′W; station 834, 38°34′N, 76°26′W; station 818, 38°18′N, 76°17′W; station 804, 38°04′N, 76°13′W; station 744, 37°44′N, 76°11′W; station 724, 37°24′N, 76°05′W; and station 707, 37°07′N, 76°07′W. Stations 858, 804, and 707 are key stations representing northern, middle, and southern Chesapeake Bay regions, respectively. During the February 2004 cruise, it was not possible to sample stations 908 and 858 due to sea ice in the northern Chesapeake Bay, and consequently, there were no water samples collected from these two stations for this cruise. In addition, no water samples were collected from station 845 during the July 2005 cruise, due to unexpected weather conditions and a change of the sampling plan.
Water samples were collected using 10-liter Niskin bottles mounted to a rosette (General Oceanics), at two depths: 1 m below the surface (designated T) and 1 m above the bottom (designated B). Middle-layer water samples (designated M) were also collected, at the pycnocline or thermocline layer, for most stations, except for station 908, where the shallow depth did not allow for the clear development of water column stratification. Upon collection, 50-ml water samples were fixed immediately with glutaraldehyde (final concentration, 1%) and stored at 4°C in the dark for Synechococcus counts, while 5-ml fixed water samples were stored at 4°C in the dark (2002 to 2003 cruises) or snap-frozen in liquid nitrogen (2004 to 2007 cruises) for bacterial and viral counts. Furthermore, subsamples of 50 ml of surface water from each station were immediately frozen (−20°C) for subsequent nutrient analysis.
Chl a and nutrient concentrations.
Water samples were size fractionated into three classes: >20 μm, 3 to 20 μm, and <3 μm. Duplicate samples (100 ml) from each station were vacuum filtered (<150 mm Hg) onto 25-mm Whatman GF/C54 filters, and chlorophyll a (Chl a) was extracted with 90% acetone for 24 h at 4°C in the dark. The Chl a concentration was determined using a Turner Designs 10-AU fluorometer. Chl a data from the March 2003 to October 2005 cruises were available for this study. The Chl a concentration in the <3-μm fraction from each water sample was assumed to be comprised of picocyanobacteria of the genus Synechococcus.
Nutrient data, including those for ammonia, nitrite plus nitrate, and phosphate, were determined using a Technicon AutoAnalyzer II at the Horn Point Analytical Services Laboratory (www.hpl.umces.edu/services/as.html). The analysis followed standard methods for chemical analysis of water and wastes proposed by the U.S. Environmental Protection Agency (U.S. EPA) (41a). Nutrient data included here are from the September 2002 to July 2006 cruises.
Direct counting of Synechococcus cells, total bacteria, and viruses.
Synechococcus cells were counted using a Zeiss Axioplan (Zeiss, Germany) epifluorescence microscope equipped with a 100-W mercury bulb (USH-102DH; Ushio Inc., Japan). Briefly, 3- to 50-ml water samples were filtered onto 0.2-μm-pore-size 25-mm black polycarbonate membrane filters (Osmonics) and counted with a green light excitation filter set (Zeiss filter set 14 [excitation band-pass filter, 510 to 560 nm, and emission long-pass filter, 590 nm]) at a magnification of ×1,000. PE-type Synechococcus cells autofluoresced in yellow to orange, while PC-type Synechococcus cells autofluoresced in red under the above settings (46, 51). At least 200 cells from 10 random fields were counted per sample.
Bacteria and viruses were enumerated following a previously described protocol (42).
MPN assay of cyanophage titers.
Synechococcus WH7803 (a member of the MC-A Synechococcus group) and CB0101 (a member of the MC-B Synechococcus group) were used as host cells for cyanophage titer measurements. They were maintained and grown in SN medium as described previously (6). The surface water samples from three key stations (stations 858, 804, and 707) from cruises in August 2003 to February 2007 were assayed. The most probable number (MPN) assay was performed following the protocol described by Suttle and Chan (40). Exponentially growing Synechococcus hosts (1.5 ml at 2 × 108 cells ml−1) were mixed with 1.5 ml of 0.22-μm-filtered water. Ten-fold serial dilutions were set up in a 96-well microtiter plate. Controls consisted of the addition of microwave-treated 0.22-μm-filtered water. Duplicate plates were set up for the same water sample, and all plates were incubated at 25°C with a 14-h–10-h light-dark cycle with illumination at 10 to 20 microeinsteins m−2 s−1. Cell lysis was monitored daily for up to 15 days, and the number of observed host cell lysis events (as determined by loss of pigments) in each dilution series was recorded. The cyanophage titers were estimated by using MPN analyzer software (no longer available). Theoretically, the detection range of this MPN assay setting was from 10 to 107 cyanophages ml−1.
Estimation of in situ contact rate of Synechococcus cells and their cyanophages.
The contact rate at which cyanophages would encounter Synechococcus cells on a daily basis was calculated using the formula R = (Sh2πwDv)VP, where Sh is the Sherwood number (dimensionless) for Synechococcus cells (1.01), w is the cell diameter (mean = 1.5 × 10−4 cm), Dv is the diffusion constant for viruses (3.456 × 10−3 cm2 day−1), and V and P are in situ cyanophage abundance (measured by MPN assay of WH7803 [see below]) and Synechococcus cell density (from direct counts), respectively (40). This formula can be simplified to R = 3.29 × 10−6 (ml day−1)VP when the above parameters are provided. The parameters are described in detail by Murray and Jackson (26).
Estimation of impact of cyanophages on Synechococcus abundance.
Assuming that the standing stock of infectious cyanophages in natural waters results from the net balance between viral production and the removal of viruses due to viral decay at a given time (40), the number of host cells (Nc) that would have to be lysed per day in order to produce the viral abundance measured by MPN assay can be calculated by using the average synechophage burst size of 83 (43), using the formula Nc = V(1 + Rd)/83. V is the cyanophage abundance measured by MPN assay (using Synechococcus WH7803 as the host), and the viral decay rates (Rd) used here were 0.03 day−1 (January, February, and March) for winter months and 0.5 day−1 for summer months (June, July, and August), with a rate of 0.12 day−1 assumed for the remaining months (11).
Phage type determination of the most abundant phage clones.
To determine the types of the most abundant cyanophage clones at the endpoint of serial dilutions from the MPN assay, PCR amplification with primers specific for the cyanomyovirus g20 gene (42) and the cyanopodovirus pol gene (43) was employed following previously described protocols (43). The phage clones were assumed to be siphoviruses if none of the above gene markers yielded a positive PCR amplification (while some phage clones have been confirmed to be siphophages, others remain to be determined).
RESULTS
Hydrographic conditions.
Since the Chesapeake Bay is a temperate estuary, its water temperature varied dramatically in different seasons (see Fig. S2, upper panel, in the supplemental material). The average surface water temperatures during the cruises ranged from 1.7°C (February 2007) to 28.3°C (August 2005). The highest surface water temperature observed was 29.4°C (station 804, August 2005), while the lowest was −0.03°C (station 858, February 2007).
The Chesapeake Bay is also a typical salt-wedge estuary, and thus its salinity increased gradually from station 908 to station 707 (see Fig. S2, middle panel, in the supplemental material). The contour plot of salinity in different seasons reflects the mixing of freshwater and oceanic water. Salinity at the nine sampling stations varied from 2.2 ppt (station 908 in March 2003 and May 2004) to 26.8 ppt (station 707, September 2002). After a prolonged drought period from 1999 to 2002, Chesapeake Bay received a large amount of precipitation through 2003 and 2004. In fact, 2003 was the wettest year in over a century (National Weather Service annual review report [http://www.nws.noaa.gov/]). This dramatic change was reflected by the freshwater discharge from the three main rivers in the Chesapeake Bay (see Fig. S2, lower panel, in the supplemental material). According to USGS monthly water resource reports (http://waterdata.usgs.gov/nwis), freshwater flow in the first 5 months of 2003 was double the amount in the same period of 2002. In 2003, the average freshwater flow was about 2-fold higher than historical July and August averages and 4-fold higher than the historical September average. The years 2003 and 2004 were featured as “deluge years” for the Chesapeake Bay, and increased freshwater flow consequently lowered the salinity throughout the Bay. The average salinity of the nine stations was 11.7 ppt in 2003 and 11.6 ppt in 2004, considerably lower than the salinities seen in the “drought years” of 2005 (13.3 ppt) and 2006 (13.9 ppt).
Nutrient concentrations (N and P) in Chesapeake Bay surface water also demonstrated significant seasonal and spatial variations (see Fig. S3 in the supplemental material). The measured total nitrogen (nitrate, nitrite, and ammonium) concentration was generally higher in the northern part of the Bay (station 858) than in the middle (station 804) and southern (station 707) parts of the Bay. The average concentrations of nitrate plus nitrite ranged from 26.2 μM (station 858) to 10.7 μM (station 804) to 2.9 μM (station 707) and appeared to have a negative linear relationship with water salinity (see Fig. S4, upper panel). In contrast, there was no obvious correlation between the ammonium or phosphorus concentration and salinity (see Fig. S4, middle and lower panels). The concentrations of nitrate and nitrite appeared to be high in the cold seasons (winter and early spring) and low in summers (June to August). Phosphorus (phosphate) concentrations were highest in the southern Bay in February 2004 and also showed peaks in July 2003, August 2003, and October 2005.
Temporal and spatial variations in Synechococcus abundance.
A temperature-driven pattern of Synechococcus abundance was obvious and annually recurrent from 2003 to 2006 (see Fig. S5, upper and middle panels, in the supplemental material). Synechococcus abundance was positively correlated with water temperature (Fig. 1) and often peaked in summer (June to August). The cell density of Synechococcus exceeded 1 million cells ml−1 in the summers of 2003, 2004, and 2006. The highest cell density (3.3 × 106 cells ml−1) was recorded at station 818 (surface water sample) in June 2006. Despite the strong salinity and nutrient (particularly nitrate and nitrite) gradients along the Bay, total Synechococcus counts remained relatively stable throughout the Bay compared to the seasonal variations. During the summer blooms, Synechococcus cells comprised up to 32% of total bacterial abundance (see Fig. S5, lower panel) and contributed, on average, 20 to 40% of total phytoplankton chlorophyll concentrations (Fig. 2). It was observed that Synechococcus spp. alone could make up to ca. 80% of the total phytoplankton chlorophyll concentration in the Bay water during the summer (station 804 in July 2005 [data not shown]). Synechococcus populations were responsible for up to 50 to 60% of total primary production in the southern stations (Wayne Coats, Smithsonian Environmental Research Center, MD, personal communication). In winter and early spring, the Synechococcus cell density decreased dramatically, to a few hundred cells per milliliter. The lowest Synechococcus count (67 cells ml−1) was recorded at station 744 (bottom water) in April 2003. Across the entire data set, the average Synechococcus cell density was 6.97 × 104 cells ml−1 for bottom waters and 3.00 × 105 cells ml−1 for surface waters. Notably, Synechococcus abundance was lower in the summer of 2005 (3-fold lower, on average) than in other summers (Fig. 2). This resulted in a lowered percent contribution of Synechococcus to total bacterial abundance (see Fig. S5, lower panel) but did not affect the contribution of Synechococcus to the total Chl a concentration (Fig. 2).
Fig. 1.
Fig. 2.
The composition of PC-enriched and PE-enriched Synechococcus varied greatly from the northern to southern parts of the Bay (see Table S1 in the supplemental material). There were pronounced seasonal as well as spatial variations in the distribution of PE- versus PC-type Synechococcus in the Chesapeake Bay. During summer blooms, the PC-type Synechococcus cells were dominant (>75%) in surface waters of the northern Bay region, and the percentage of PE-type cells gradually increased from the northern to the southern Bay (see Table S1).
In the winter, PE-type Synechococcus cells were predominant throughout the Bay and comprised up to 100% of the picocyanobacterial community in the middle and southern Bay regions. The ratio of PC-type to PE-type cells appeared to be influenced by the salinity gradient in summer months (see Fig. S6 in the supplemental material). A larger proportion of PE-type Synechococcus cells was observed in the 2005 and 2006 summers than in the summer of 2004. The percentage of PE-type Synechococcus cells appeared to increase from surface to bottom waters in summer (see Table S1).
Synechococcus phage titers and distributions.
The titers of cyanophages infective to Synechococcus WH7803 ranged from 105 to 6.2 × 105 infectious units ml−1 (Table 1 and Fig. 3). In parallel with total Synechococcus counts, WH7803 phage titers were high in summer and low in winter, and they exceeded the total Synechococcus cell abundance in some cases. The middle Bay region (station 804) had higher cyanophage titers, with an annual average of 6.9 × 104 ml−1, than the northern Bay (2.6 × 104 ml−1) and southern Bay (4.6 × 104 ml−1) regions. The overall mean infectious synechophage titer in the Bay was 4.7 × 104 ml−1, which is 6-fold lower than the overall mean Synechococcus host abundance (3.0 × 105 ml−1).
Table 1.
| Date and station | No. of Synechococcus cells (104 ml−1) | WH7803 phage MPN (104 ml−1) | CB0101 phage MPN (102 ml−1) | Ratio of WH7803 phage titer to CB0101 phage titer | Most abundant WH7803 phage typea | % Cyanophages among total virus-like particlesb | % Lysed Synechococcus cellsc |
|---|---|---|---|---|---|---|---|
| July 2004 | |||||||
| 858 | 123 | 0.09 | 0.72 | 13 | Podo | <0.1 | <0.1 |
| 804 | 144 | 62.1 | 3.1 | 1,980 | Sipho | 2.7 | 0.8 |
| 707 | 146 | 0.01 | ND | NA | Podo | <0.1 | <0.1 |
| Mean | 138 | 20.7 | 1.93 | 1,070 | NA | 0.9 | 0.3 |
| August 2004 | |||||||
| 858 | 20.4 | 0.02 | ND | NA | Sipho? | <0.1 | <0.1 |
| 804 | 56.2 | 0.15 | ND | NA | Podo | <0.1 | <0.1 |
| 707 | 28.9 | 0.37 | 0.1 | 335 | Podo | <0.1 | <0.1 |
| Mean | 35.2 | 0.18 | 0.04 | 487 | NA | <0.1 | <0.1 |
| July 2005 | |||||||
| 858 | 96.3 | 12.3 | 21.9 | 56 | Myo | 1.5 | 2.3 |
| 804 | 79.8 | 23.4 | 0.43 | 5,440 | Sipho? | 2.3 | 0.5 |
| 707 | 48.4 | 17 | 0.91 | 1,860 | Myo | 1.5 | 0.6 |
| Mean | 45.9 | 17.5 | 7.74 | 227 | NA | 1.8 | 1.1 |
| August 2005 | |||||||
| 858 | 2.45 | 23.4 | 7.13 | 328 | Myo | 1.3 | 17.2 |
| 804 | 4.55 | 5.42 | 0.02 | 27,100 | Myo | 0.2 | 2.2 |
| 707 | 8.20 | 46.2 | 0.84 | 5,500 | Podo | 2.4 | 10.2 |
| Mean | 5.07 | 25 | 2.66 | 939 | NA | 1.3 | 9.9 |
| Jun 2006 | |||||||
| 858 | 23.7 | 45.9 | 0.26 | 17,700 | Myo | 0.3 | 3.5 |
| 804 | 108 | 54.2 | 154 | 35 | Myo | 0.5 | 0.9 |
| 707 | 61.6 | 9.60 | 2.53 | 379 | Sipho | 0.2 | 0.3 |
| Mean | 64.4 | 36.6 | 52.3 | 70 | NA | 0.3 | 1.6 |
| July 2006 | |||||||
| 858 | 101 | 15.5 | 4.97 | 312 | Myo | 0.6 | 0.3 |
| 804 | 139 | 34.2 | 70.3 | 49 | Myo | 0.5 | 0.4 |
| 707 | 93.8 | 1.92 | 0.06 | 3,200 | Sipho | 0.1 | <0.1 |
| Mean | 111 | 17.2 | 2,510 | 69 | NA | 0.4 | 0.35 |
a
Cyanomyoviruses and cyanopodoviruses were detected by a PCR method (see Materials and Methods). Siphoviruses with question marks need further confirmation by either PCR-sequencing or transmission electron microscopy observation.
b
Ratio of sum of WH7803 and CB0101 phage MPN abundances to total virus-like particle counts.
c
Calculated based on WH7803 phage MPN abundance.
d
ND, not detectable; NA, not applicable.
Fig. 3.
The phage titers measured with Synechococcus strain CB0101 (a strain isolated from Chesapeake Bay [6]) were remarkably lower than WH7803 phage titers, with no phages detected in nearly half of the water samples (Fig. 3). In general, WH7803 phage titers were several-hundredfold higher than CB0101 phage titers in summer (Table 1). Despite their low abundance, CB0101 phage titers showed a distribution pattern similar to that for WH7803 phage titers, with high titers in summer and low titers in winter.
Together, WH7803 and CB0101 phage titers could contribute more than 2% of the total viral abundance in a few cases, but in general, they were below 2% of total viral counts (Table 1).
Impact of cyanophages on Synechococcus abundance.
Based on synechophage titers obtained using the WH7803-cyanophage system, a strong correlation between cyanophage abundance and host cell density was seen for 2005 and 2006; however, such a relationship was not obvious in 2004 (Fig. 4). High phage-host contact rates were often seen during summer Synechococcus blooms, when each Synechococcus cell could encounter infectious cyanophages twice a day (i.e., 204% contact rate) (Fig. 5). In contrast, less than 0.1% of Synechococcus cells collided with infectious cyanophages each day during the winter season (Fig. 5). Phage-host contact rates also varied dramatically across the Bay. For example, a range of contact rates of 0.03 to 204% was seen from the southern to middle regions of the Bay in July 2004. Overall, average per-day phage-host contact rates were 32.2, 16.9, and 13.9% for the middle, northern, and southern regions of the Bay, respectively.
Fig. 4.
Fig. 5.
The percentage of Synechococcus cells lysed by viral infection each day (infection rate) followed a trend similar to that for the contact rate (Fig. 5 and Table 1). Overall, the infection rate varied from 0.7 to 1.4% from the southern to the northern Bay. High lysis rates were often seen in the summer months. About 1 to 10% of Synechococcus cell abundance was lost to cyanophage infection each day in the summer of 2005. Synechococcus cell lysis rates were 0.35 to 1.6% in the summer of 2006 and <0.8% in the summer of 2004.
Most abundant Synechococcus phage types.
Three different types of cyanophages (myo-, podo-, and siphoviruses) could be detected in high abundance (>105 ml−1) in the summer samples (Table 1). The frequency of each phage type varied in different years. The most abundant Synechococcus WH7803 phage type was either podovirus or siphovirus in summer 2004, but no myoviruses could be detected by PCR with g20 gene-specific primers. Cyanomyoviruses were the dominant morphotype in the summer of 2005 and 2006, as 67% of MPN endpoint lysates were positive for the cyanomyovirus g20 gene by PCR amplification (Table 1). Overall, cyanomyoviruses accounted for 44% (8 of 18 samples) of the most abundant phage clones.
DISCUSSION
Our multiyear survey showed that total Synechococcus counts and cyanophage titers covary and exhibit strong seasonal patterns in Chesapeake Bay. A strong correlation between water temperature and Synechococcus abundance was observed in the Bay over the 4-year survey period (Fig. 1). The temperature-driven annual pattern of Synechococcus abundance has been observed in temperate coastal waters (23, 45) and estuarine waters (1, 3, 28). Warm temperature perhaps favors the rapid growth of Synechococcus in the Bay. Synechococcus counts in the Chesapeake Bay often exceeded 106 cells ml−1 during summer months. The annual average Synechococcus count in the Bay is 3 × 105 cells ml−1 (overall average based on the entire data set), which is 10- to 100-fold higher than those reported for open oceans (typically 103 to 104 cells ml−1) (17) and North Atlantic coastal water (23, 45). The abundance of Synechococcus in Chesapeake Bay is comparable with those reported for San Francisco Bay (28) and subtropic Florida Bay estuarine ecosystems (27, 34).
No clear correlation between Synechococcus abundance and the concentrations of nutrients (nitrate plus nitrite, ammonia, and phosphate) was observed in the Chesapeake Bay. The nutrient concentrations in the Bay are usually high during the winter-spring period, mainly due to strong riverine inputs (20). It is known that Synechococcus can utilize various nitrogen sources for growth (8, 24, 31, 46). High nutrient levels in the Bay and the ability to utilize various sources of nitrogen may explain the weak relationship between Synechococcus abundance and the levels of inorganic nutrients.
Although salinity had little impact on the spatial variation of total Synechococcus counts, the distribution of PC- versus PE-type Synechococcus was affected by the salinity gradient. PC-type Synechococcus cells were dominant in the northern Bay, while PE-type Synechococcus cells were more abundant from the middle to the southern Bay. A positive correlation was seen between the percentage of PE-type Synechococcus cells and salinity along a north-south transect in the Bay, indicating that salinity could be an important factor influencing the distribution of PE- and PC-type Synechococcus cells in the Chesapeake Bay. Interannual variation in the percentage of PE-type Synechococcus cells also appeared to correlate with salinity changes. In the deluge year of 2004, the percentage of PE-type cells in the summer surface water was ca. 38% in the middle Bay but climbed to 64 to 88% during July and August in the drought year of 2005.
Cyanophages infecting Synechococcus WH7803 could be detected in all seasons and in different locations in the Chesapeake Bay, indicating that they are ubiquitous and persistent members of Chesapeake Bay microbial communities. The high abundance of Synechococcus phages (commonly >105 ml−1) detected during summertime suggested that they are also important members within the Chesapeake Bay virioplankton. A recent metagenomic analysis of Chesapeake Bay virioplankton also revealed a large proportion of cyanophage sequences in the late summer (September 2002) sample (4). The dynamic variation in Synechococcus phage titers indicates that they are active pathogens of host Synechococcus populations. Synechococcus WH7803 is sensitive to phage infection and has been used to titrate cyanophages in many studies (19, 23, 25, 38, 40). Nonetheless, the phage titers obtained using WH7803 alone cannot represent all cyanophages infecting all Synechococcus spp. present in the Bay and can be interpreted here only as underestimates of the total cyanophage abundance.
The tight covariation between Synechococcus abundance and cyanophage titers in the Chesapeake Bay indicates that cyanophages likely play a role in regulating the abundance and productivity of coexisting Synechococcus populations. The average daily lysis rate for Synechococcus populations in summer was 2.2%, which agrees with previously reported transmission electron microscopy observations (0.8 to 2.8%) of natural Synechococcus populations (35). The average Synechococcus infection rate (considering all seasons) of cyanophages was 0.96%, which is lower than that reported for Gulf of Mexico waters (40) but higher than that reported for Woods Hole Harbor water (45).
The percent infected Synechococcus cells varied greatly in summers from 2004 to 2006. In the high-rainfall summer of 2004, the contact rate between hosts and infectious cyanophages was remarkably high (up to 204% day−1 at station 804). Correspondingly, infection rates of <1% of Synechococcus cells were necessary to produce the observed phage titers. This observation is consistent with previous reports from Woods Hole Harbor (45). In August 2005, 77% of Synechococcus cells were contacted by cyanophages on a daily basis, with a 17.2% Synechococcus lysis rate. At the same time, the Synechococcus cell abundance was ca. 2.5 × 104 cells ml−1. The ratio of virus to Synechococcus bacteria was ca. 10, which was the highest one observed in this study. Notably, the overall cyanophage infection rate appeared to be high throughout the Bay in August 2005, as 10.2% and 2.2% of Synechococcus cells in the lower and middle regions of the Bay, respectively, were infected. The higher infection rates of cyanophages concurred with lowered Synechococcus host densities in these summers and were likely due to host population composition variations, shaped largely by the hydrological conditions. More PE-type Synechococcus cells persisted in summer 2005 than in summer 2004, and these might be more susceptible to cyanophage infection, particularly to polyvalent cyanomyovirus infection (Table 1). In contrast, podoviruses and siphoviruses appeared to be the predominant cyanophage types during summer 2004, while myoviruses prevailed in summer 2005. Cyanomyoviruses are believed to have a broad host range, while podo- and siphoviruses are usually strain specific (5, 38, 41, 45). This phenotypic difference among distinct groups of cyanophages may account for the observed differences in infection rates between years. Since cyanomyoviruses are able to infect a broader range of host cells, they may have had a greater impact on overall host population abundance than host-specific cyanophages, while cyanopodoviruses and cyanosiphoviruses may play more specific roles in shaping host population structure by scavenging those sensitive cells.
In conclusion, highly abundant Synechococcus cells and their phages were observed repeatedly in summer waters in the Chesapeake Bay, indicating that they are important microbial components in this eutrophic estuary. Seasonal covariation between Synechococcus and cyanophage abundances suggests that these organisms have active and dynamic interactions in the Bay. The cyanophages could infect a substantial portion of Synechococcus cells during summer blooms, indicating that they may play important roles in regulating the host population and subsequently influence the carbon fixation and nutrient flow in the estuarine ecosystem. In a broader sense, viral infection-induced cell lysis may contribute a substantial dissolved organic matter (DOM) pool to world oceans, short-circuit the nutrient and energy flow via a viral shunt in the microbial loop, and consequently influence physiochemical processes through the microbial carbon pump (MCP) (14). In the Chesapeake Bay, we found that the impacts of cyanophages on Synechococcus mortality varied under different conditions, being higher in the drought years but lower in the deluge years. We speculated that the variance in environmental gradients might influence the host composition and consequently affect the viral impacts on host population. To that end, establishing a specific host-virus system (for instance, the Synechococcus-cyanophage system presented here) would be an ideal case to illustrate the emergent impact of viral infection on the biogeochemistry of the Bay.
ACKNOWLEDGMENTS
We gratefully thank all the people who participated in and contributed to the Microbial Observatory for Virioplankton Ecology in the Chesapeake Bay (MOVE) project (2002 to 2007). The MOVErs were D. Wayne Coats, Kurt E. Williamson, Danielle M. Winget, Rebekah R. Helton, Shannon Williamson, Shelly Bench, Sharath Srinivasiah, Jinjun Kan, Sean Cooney, Yvan Bettarel, Yoanna Eissler, and Matthew Feinberg. We thank the crews of the R/V Cape Henlopen and R/V Hugh R. Sharp for assisting with sample collection.
This work was supported by grants from the National Science Foundation (OCE-9730602, OCE-0049098, and MCB-0132070).
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Published In
Applied and Environmental Microbiology
Volume 77 • Number 21 • 1 November 2011
Pages: 7459 - 7468
PubMed: 21821760
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© 2011 American Society for Microbiology.
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Received: 7 February 2011
Accepted: 31 July 2011
Published online: 25 October 2011
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