Viral distribution and life strategies in the bach dang estuary, vietnam

Microb Ecol (2011) 62:143–154 DOI 10.1007/s00248-011-9835-6 MICROBIOLOGY OF AQUATIC SYSTEMS Viral Distribution and Life Strategies in the Bach Dang Estuary, Vietnam Yvan Bettarel & Thierry Bouvier & Martin Agis & Corinne Bouvier & Thuoc Van Chu & Marine Combe & Xavier Mari & Minh Ngoc Nghiem & Thuy Thanh Nguyen & Thu The Pham & Olivier Pringault & Emma Rochelle-Newall & Jean-Pascal Torréton & Huy Quang Tran Received: 24 November 2010 / Accepted: 17 February 2011 / Published online: March 2011 # Springer Science+Business Media, LLC 2011 Abstract Although the structure and dynamics of planktonic viruses in freshwater and seawater environments are relatively well documented, little is known about the occurrence and activity of these viruses in estuaries, especially in the tropics Viral abundance, life strategies, and morphotype distribution were examined in the Bach Dang Estuary (Vietnam) during the dry season in 2009 The abundance of both viruses and their prokaryotic hosts decreased significantly from upstream to downstream, probably as the result of nutrient dilution and osmotic stress faced by the freshwater communities The antibiotic mitomycin-C revealed that the fraction of lysogenic cells was substantially higher in the lower seawater part of the estuary (max 27.1%) than in the upper freshwater area where no inducible lysogens were observed The question of whether there is a massive, continuous induction of Y Bettarel (*) : T Bouvier : M Agis : C Bouvier : M Combe : X Mari : O Pringault : J.-P Torréton UMR 5119, ECOSYM, Montpellier University, CNRS, IRD, IFREMER, Montpellier, France e-mail: yvan.bettarel@ird.fr T Van Chu : T T Pham Institute of Marine Environment and Resources (IMER), Hai Phong, Vietnam M N Nghiem Institute of Biotechnology (IBT), Hanoi, Vietnam T T Nguyen : H Q Tran National Institute of Hygiene and Epidemiology (NIHE), Hanoi, Vietnam E Rochelle-Newall UMR 7818 BIOEMCO, IRD, Paris, France marine lysogens caused by the mixing with freshwater is considered Conversely, the production of lytic viruses declined as salinity increased, indicating a spatial succession of viral life strategies in this tropical estuary Icosahedral tailless viruses with capsids smaller than 60 nm dominated the viral assemblage throughout the estuary (63.0% to 72.1% of the total viral counts), and their distribution was positively correlated with that of viral lytic production Interestingly, the gamma-proteobacteria explained a significant portion of the variance in the 90 nm) was explained by the beta-proteobacteria Overall, these results support the view that the environment, through selection mechanisms, probably shapes the structure of the prokaryotic community This might be in turn a source of selection for the virioplankton community via specific affiliation favoring particular morphotypes and life strategies Introduction Viruses form a ubiquitous, dynamic compartment in both seawater and freshwater environments where they fulfill numerous biogeochemical and ecological functions [49] They are now considered as major players in the ecological balance of aquatic ecosystems Primarily targeting prokaryotes, planktonic viruses interact with their hosts in two main different ways: lytic and lysogenic infection cycles [53] and, more sporadically, through chronic cycles or pseudolysogeny [57] Although viruses are the most abundant and probably the most diverse biological entities on earth [47], relatively little is known about the patterns of viral distribution within 144 and between the main types of aquatic habitat During the last two decades, much attention has been paid to marine coastal and oceanic waters and, to a lesser extent, lacustrine freshwater from a virio-ecological perspective, especially in temperate zones However, few studies have been carried out in transition zones such as estuaries, particularly in Asia Estuaries are interesting areas for research because they are among the most productive and exploited aquatic habitats They often have strong eutrophication and salinity gradients as a result of the dilution of nutrient-rich river water by seawater Studying viral distribution along such steep gradients is of great interest for microbial ecologists because the mixing front between marine tidal water and outflowing river water is an area of dramatic changes which can trigger important physiological, genetic, and ecological shifts in their microbial hosts [11, 24] Previous studies on estuarine virioplankton communities have shown that abundance is typically correlated with the distribution of prokaryotes for which they can be a major cause of mortality [1, 2, 16] Sometimes conflicting correlations have been found between viral abundance and some physical and chemical parameters such as salinity, chlorophyll a, suspended material or nutrient concentrations, and temperature [4, 23] However, no clear pattern has emerged as estuaries are often characterized by a unique, complex combination of hydrodynamic, trophic, and thermal conditions Recent reports have shown that viral diversity and richness observed in aquatic biomes can change rapidly over time and space [3, 6, 19, 43, 59] In Chesapeake Bay, for example, the virioplankton community structure, as inferred from PFGE and RAPD-PCR analyses, was reported to exhibit seasonally and spatially dynamic patterns of diversity [22, 60] However, this was not the case in the Charente Estuary (France) where the genetic and morphological structure of the virioplankton community was relatively stable [5] Unfortunately, no other reports of viral diversity in estuaries could be found to explain why the structure of a viral community can remain stable in environments where biophysicochemical characteristics are so heterogeneous Moreover, the distribution of viral life strategies along estuarine gradients is still unknown Clearly, further studies are needed to provide a better understanding of the factors that govern viral abundance, proliferation, and diversity in all estuarine systems This study examined the distribution of viruses, the prevalence of lytic versus lysogenic strategies, and the morphological composition of the virioplankton community It was conducted in the Bach Dang Estuary, one of the main tributaries in the Red River Delta, Vietnam, a tropical region with rapid population growth and the main source of agricultural produce for North Vietnam Y Bettarel et al [62] It also assessed whether the variability of these viral parameters could be explained by the local environmental specificities (salinity, nutrients, temperature, and dissolved organic carbon) and/or the distribution and the phylogenetic composition of their prokaryotic hosts Material and Methods Description of the Study Site Samples were collected on March 12, 13, and 15, 2009, between 07:00 and 10:00 during neap tides, throughout the salinity gradient of the Bach Dang Estuary, one of the main tributaries of the Red River in Vietnam (Fig 1) The Red River is characterized by high human pressure and by the transport of vast amounts of fine sediment during seasonal monsoons [42, 62] Samples were taken from 15 stations along the estuary over a salinity gradient of to 31 (Table 1) At each station (nos 1–15, see geographical coordinates in Table 1), samples were collected in subsurface water (1.5 m depth) using a Niskin bottle Duplicate samples were analyzed for nutrient and chlorophyll a (Chl) content, as well as for bacterial and viral parameters Samples for dissolved inorganic nutrient measurements (N-NO2, N-NO3, N-NH4, P-PO4) were filtered through precombusted Whatman GF/F fiberglass filters, stored at −20°C and analyzed according to Eaton et al [18] Chl concentrations were determined by fluorometry after filtration onto Whatman GF/F filters and methanol extraction [24] Dissolved organic carbon (DOC) analyses were performed on 30 mL subsamples filtered onto precombusted GF/F filters and stored in precombusted (450°C, overnight) 40 mL glass vials with Teflon stoppers, with 35 μL 85% phosphoric acid (H3PO4) Samples were stored in the dark until analysis using a Shimadzu TOC VCPH analyzer Potassium phthalate was used as a calibration standard, and certified reference materials (Hansell Laboratory, University of Miami) were also used to verify the instrument Salinity and temperature were measured in situ using a CTD probe (Seabird SBE 19+) Counts of Viruses and Prokaryotes Water samples were fixed with 0.02 μm filtered buffered formaldehyde (final concentration 2% v/v) after sampling, immediately flash frozen in liquid nitrogen, and stored at −80°C prior to counting The number of viruses and prokaryotes in the duplicate samples of 0.3–0.8 mL was determined after retention on 0.02 μm pore size membranes (Anodisc) and staining with SYBR Gold fluorochrome (Molecular Probes, Europe, Leiden, the Netherlands) as described in detail by Patel et al [36] Viral Distribution and Life Strategies 145 Figure Map of the Bach Dang River Estuary (Vietnam) and location of the fifteen sampling stations, March 2009 km Table Geographical coordinates and physicochemical parameters of the sampled stations in the estuary of the Bach Dang River, Vietnam, March 2009 Station nos Latitude Longitude 1ab 2b 3ab 5ab 8ab 9ab 10b 11 12ab 13 14 20°52.729 20°56.489 20°56.525 20°50.813 20°49.700 20°49.940 20°49.819 20°48.700 20°45.791 20°47.396 20°45.174 20°43.466 20°41.295 20°43.299 15ab 20°40.743 N N N N N N N N N N N N N N N Salinity Temp (°C) Chl a (μg L−1) E E E E E E E E E E E E E E 0.1 1.4 4.1 5.5 9.2 10.5 10.5 15.0 21.0 26.1 26.1 27.8 28.8 28.9 21.2 21.3 21.7 21.2 21.2 21.3 21.2 20.4 19.9 20.8 20.8 19.7 20.5 20.7 1.8 0.2 9.4 4.0 1.9 2.0 1.5 2.8 3.4 2.6 2.2 2.1 1.6 1.7 106°59.641 E 30.6 19.9 0.8 106°39.758 106°46.224 106°46.338 106°46.218 106°48.450 106°47.480 106°49.168 106°50.409 106°51.386 106°55.082 106°56.675 106°53.232 106°54.972 106°58.144 N-NO2 (μM) N-NO3 (μM) N-NH4 (μM) P-PO4 (μM) 113 109 132 117 110 120 140 115 105 117 122 111 93 109 1.8 1.6 2.3 2.5 2.0 1.7 2.1 1.5 1.2 1.6 1.2 1.3 1.1 1.2 14.9 20.4 15.6 16.1 11.5 10.3 13.7 9.9 6.4 12.8 10.7 7.0 9.8 9.3 9.1 9.4 13.4 11.5 10.1 8.7 12.7 8.8 8.2 6.9 5.4 2.7 5.4 5.5 1.1 1.2 1.3 1.0 0.9 0.9 1.1 0.7 0.5 1.2 1.1 0.4 0.9 0.5 97 0.8 9.1 4.7 0.6 DOC (μM-C) a stations where the viral lytic production and the fraction of lysogenic cells were estimated b stations where the distribution of viral morphotypes and the phylogenetic composition of the prokaryotic communities were estimated 146 Examination of Viral Morphotypes Planktonic viruses were observed using transmission electron microscopy (TEM) Viruses from 500 μL aliquots of formalinfixed samples were harvested by repeated ultracentrifugation of 50 μL onto grids (400 mesh Cu electron microscope grids with carbon coated Formvar film) using an A-100/30 rotor in an air-driven ultracentrifuge (Airfuge®, Beckman) at 105,000×g for 70 The grids were then stained for 30 s with uranyl acetate (2%, w/w), and viruses were examined and measured using a JEOL 1200EX TEM operated at 80 kV and magnification from ×20,000 to ×100,000 Three morphotypes were distinguished for shape classification of tailed viruses (Caudovirales) Tailed viruses with isometric heads and long noncontractile tails were considered to be siphoviruses Tailed viruses with isometric heads and contractile tails (presence of a neck) were considered to be myoviruses Tailed viruses with isometric heads and short tails were considered to be podoviruses (see Fig 4) The distribution of tailless icosahedral viruses in size classes 90 nm was also determined Fraction of Lysogenic Cells The fraction of lysogenic cells (FLC) was determined by the induction of prophages using mitomycin-C [52] Mitomycin-C was added to samples (final concentration μg mL−1) in 20 mL sterile serum bottles and untreated samples served as controls Samples were incubated at in situ temperature; duplicate subsamples were taken with syringes at the start of incubation (t0) and after 12 h (t12h) and fixed with 0.02 μm filtered buffered formaldehyde (2% final concentration) for viral and bacterial counts (see above) The FLC was estimated from viral abundances in mitomycinC treated (VAm) versus control (VAc) incubations, as well as bacterial abundance (BAt0) and burst size (BSt0): FLC ẳ 100 ẵVAm VAc Þ=ðBSt0 À BAt0 ފ [52] The burst size chosen in this study (BS=24) was the mean value for aquatic environments calculated by Wommack and Colwell [61] and Parada et al [35] Viral Lytic Production Viral production was determined using the dilution technique described by Wilhelm et al [56] Fifty milliliters of duplicate subsamples was filtered onto a 47-mm diameter, 0.2-μm pore size polycarbonate membrane at low pressure (