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Biosysteras
Diversity
Biosystems
Diversity
ISSN 2519-8513 (Print) ISSN 2520-2529 (Online) Biosyst. Divers., 2021, 29(2), 151-159 doi: 10.15421/012120
Received 11.04.2021 Received in revised form
07.05.2021 Accepted 09.05.2021
Virioplankton as an important component of plankton in the Volga Reservoirs
A. I. Kopylov, E. A. Zabotkina
Papanin Institute for Biology oflnland Waters, Russian Academy of Sciences, Borok, Russia
Kopylov, A I, & Zabotkina, E A (2021). Virioplankton. as an important component of plankton in the Volga Reservoirs. Biosystems Diversity, 29(2), 151-159. doi:10.15421/012120
The distribution of virioplankton, abundance and production, frequency of visibly infected cells of heterotrophic bacteria and autotrophic picocyanobacteria and their virus-induced mortality have been studied in mesotrophic and eutrophic reservoirs of the Upper and Middle Volga (Ivankovo, Uglich, Rybinsk, Gorky, Cheboksary, and Sheksna reservoirs). The abundance of planktonic viruses (VA) is on average by 4.6 ± 1.2 times greater than the abundance of bacterioplankton (BA). The distribution of VA in the Volga reservoirs was largely determined by the distribution of BA and heterotrophic bacterioplankton production (PB). There was a positive correlation between VA and BA and between VA and PB. In addition, BA and VA were both positively correlated with primary production of phytoplankton. Viral particles of 60 to 100 pm in size dominated in the phytoplankton composition. A large number of bacteria and picocyanobacteria with viruses attached to the surface of their cells were found in the reservoirs. Viruses as the most numerous component of plankton make a significant contribution to the formation of the planktonic microbial community biomass. The number of phages inside infected cells of bacteria and picocyanobacteria reached 74-109 phages/cell. Easily digestible organic matter, which entered the aquatic environment as a result of viral lysis of bacteria and picocyanobacteria, could be an additional source of carbon for living bacteria. The results of long-term studies indicate a significant role of viruses in functioning of planktonic microbial communities in the Volga reservoirs.
Keywords: viruses; viral lysis; heterotrophic bacteria; picocyanobacteria; microbial communities; freshwater ecosystems.
nary development (Weinbauer & Rassoulzadegan, 2004; Zhang & Gui, 2018). Studies on the role of viruses in the structure and functioning of planktonic communities in reservoirs of the Upper and Middle Volga have been carried out since 2005 to the present (Kopylov et al., 2007, 2011a, 2011b, 2013a, 2013b, 2016). In this paper, we analyze the results of long-term studies of the abundance and production of free viruses, the abundance of viruses attached to bacteria, the size of viral particles, frequencies of visibly infected cells, and virus-induced mortality of heterotrophic bacteria and picocyanobacteria in six reservoirs of the Upper and Middle Volga basin. A quantitative assessment of the role of viruses in carbon fluxes in planktonic food webs of the reservoirs has been performed.
Russian Academy of Sciences, Borok, 109, Yaroslavskaya obi., 152742, Russia. Tel.: +79-038-269-011. E-mail: kopylov@ibiw. ru
Introduction
Research over the past three decades has firmly established that viruses are the most abundant and diverse biological entities (Bratbak & Hel-dal, 2000; Wommack & Colwell, 2000; Weinbauer, 2004), thereby forming an integral component of microbial food web (viruses, bacteria, pico-phytoplankton, protozoa) in a great variety of aquatic environments (Bratbak et al., 1994; Clasen et al., 2008; Wilhelm & Matteson, 2008). Viral lysis plays fundamental roles in cycling nutrients and organic matter (Middelboe et al., 2008; Staniewsski & Short, 2014), structuring the microbial food web dynamics and regulating carbon flow (Fuhrman, 1999; Bouvy et al., 2011; Breitbart, 2012), governing microbial diversity (Weinbauer & Rassoulzadegan, 2004; Keshri et al., 2017; Zheng et al., 2020). Bacteriophages prevail in virioplankton in many aquatic ecosystems, including reservoirs (Peduzzi & Schiemer, 2004; Ram et al., 2005; Sime-Ngan-do, 2014; Hanson et al., 2017). It has been reported that viruses cause the mortality of a significant part of the marine and freshwater bacterioplankton (to 40-60% of the bacterial production) (Wommack & Colwell, 2000; Suttle, 2005, 2007; Wilhelm & Matteson, 2008; Ram et al., 2011) and, at times, can match grazing by bacterivorous protists as a source of bacterial mortality (Fischer & Velimirov, 2002; Peduzzi & Schiemer, 2004). Viruses cause the mortality of a significant part of the marine bacterioplankton (to 40-60% of the total mortality) (Fuhrman, 1999; Wommack & Colwell, 2000; Weinbauer, 2004; Suttle, 2005, 2007). Studies have shown that viruses infecting cyanobacteria (cyanophages) can be major players affecting the mortality and structure of cyanobacterial communities (Mann, 2003; Zong et al., 2018). In pelagic food webs carbon content of bacteria and picocyanobacteria can be either transferred to the upper trophic levels when consumed by protozoa and multicellular filter feeders or, alternatively, contribute to the dissolved organic matter stocks as a result of viral-induced cell lysis (Ram et al., 2015; Vaqué et al., 2017). In addition, viruses mediate the gene transfer within and between species by transduction (Jiang & Paul, 1998; Sime-Ngando, 2014). It has been hypothesized that viral genes and viral activity generate the genetic variability of prokaryotic organisms and are a driving force for ecological functioning and evolutio-
Materials and methods
The studies were conducted at the end of July-beginning of September 2005-2010 in six mesotrophic and eutrophic reservoirs of the Volga River and Volga-Baltic waterway (hereinafter referred to as "Volga reservoirs"): Ivankovo (at 11 stations), Uglich (10 stations), Rybinsk (34 stations), Gorky (16 stations), Cheboksary (20 stations), and Sheksna (12 stations). The abundance and production of viruses, bacteria, and picocyano-bacteria were determined in integrated water samples.
Planktonic viral particles were counted by epifluorescence microscopy using the SYBR Green I fluorochrome (Thermo Fisher Sci, USA) and aluminum oxide Anodisc filters (Wathman, GE Healthcare, USA) with a pore diameter of 0.02 pm (Noble & Fuhrman, 1998); heterotrophic bacteria were enumerated using DAPI (Thermo Fisher Sci, USA) as a fluorochrome and black nuclear filters with a pore diameter of 0.2 pm (Nuc-leopore, GE Healthcare, USA) (Porter & Feig, 1980). Picocyanobacteria were enumerated by epifluorescence microscopy according to autofluorescence of phytopigments of picoautotrophic cells (Maclsaac & Stockner, 1993). Filters were examined under an Olympus BX51 epifluorescent microscope (Olympus, Japan) using Cell-F image analysis software at x1000 magnification. For each water sample, two filters were analyzed; counts yielded a minimum of 600 viruses, 400 bacteria and 400 picocya-
nobacteria The carbon content in wet bacterial biomass was calculated using the equation describing the relationship between the cell volume (V, ^m3) and carbon content (C, fg C/cells): C = 120 x V072 (Norland, 1993).
The carbon content per one viral particle was taken as 10-10 ^g C (Gonzalez & Suttle, 1993). It was assumed that the carbon content constituted 16.5% of the wet picocyanobacteria biomass (Jochem, 1988).
The production of bacteria was determined by the 14C method (Ro-manenko & Kuznetsov, 1974) and dilution method (Landry & Hassett, 1982). The production of picocyanobacteria was estimated using the frequency of dividing cells of picocyanobacteria (McDuff & Chisholm, 1982). The primary production of phytoplankton was determined by the 14C method (Romanenko & Kuznetsov, 1974). Grazing rates of hetero-trophic nanoflagellates on bacteria were measured using the method of fluorescently labelled bacteria (Simek et al., 2001). Each experiment was replicated twice or thrice.
Transmission electron microscopy was used to estimate the frequency of visibly infected cells (FVIC, % of the total bacterial abundance) and the mean number of fully matured phages in infected bacteria (i.e., burst size (BS), particles/cell). For electron microscopy, viruses and bacteria were centrifuged at 100,000 g for 1 hour using an OPTIMA L-90k ultracentrifuge with a 45 Ti rotor (Beckman Coulter, USA) on Pioloform/car-bon-coated 400-mesh nickel grids (SPI, USA). The grids were analyzed under a JEM 1011 electron microscope (Jeol, Japan) at x50,000-150,000 magnification. At least 800 bacterial cells and 600 viruses were analyzed for each grid. Mature phages become clearly visible in the host cell only at the end of the latent period immediately prior to cell lysis. The fraction of all infected cells in bacterioplankton (FIC, % of the total abundance of bacteria) and viral-mediated mortality of bacteria (VMB, % of bacterioplankton production) were calculated according to the equations (Binder, 1999).
The fraction of all infected picocyanobacterial cells (FIC, of the total abundance of bacteria) and fraction of picocyanobacterial mortality due to
viral lysis (FMVL, % of the daily production of picocyanobacteria) were calculated according to the equation (Mann, 2003). The rate of virus-induced mortality of bacteria or picocyanobacteria (VIM) expressed as cells/(mL x day), or as mg C/(m3 x day) was calculated using the equation: VIM = VMB x PBAC, where PBAC is bacterioplankton production, or VIM = FMVL x PPC, where PPC is picocyanobacterial production. The virioplankton production (PV) was determined as the product of BS by VIM (cells/(mL x day)) (Noble & Steward, 2001). The viral turnover time (VTT) was determined by dividing the abundance of free viruses by their production.
The rate of easily oxidizable organic matter release from the lysed bacterial and picocyanobacterial cells into an aquatic environment was calculated as the difference between VIM (mg C/(m3 x day)) and PV (mg C/(m3 x day)). The values that were obtained are overestimates, since values of energy consumption of viruses for the synthesis of capsid proteins and replication of nucleic acids were not taken into account in our calculations because these data are not available in the literature.
Results
Characteristics of abiotic factors, abundance, biomass of bacteria and picocyanobacteria, phytoplankton primary production and bacterioplankton production. In 2005-2007, the average water temperature for the study period ranged within 19.9-21.4 °C in August and 16.6-17.2 °C at the beginning of September (Table 1). In July 2010, the water temperature in the Gorky and Cheboksary reservoirs increased to record values that were higher than the water heating under the normal thermal regime by 8.0-8.5 °C. The main reason for the anomalously high air temperature in the central regions of European Russia, and, as a result, the high water temperature in the Volga reservoirs was the blocking anticyclone of an unusual intensity and duration of the existence (55 days).
Table 1
Depth (D), transparency (Tr) and temperature (T) of water, abundance of bacteria (NB) and biomass (BB), phytoplankton primary production (Pphy) and bacterioplankton production (PB) of reservoirs (on average for a reservoir)
Reservoir Parameters D, m Tr, cm T, °C Nb, 106 cells/mL Bb, mg C/m3 Pphy, mg C/(m3 x day) PB mg C/(m3 x day)
Ivankovo x ± SD 8.8 ± 1.4 90 ± 8 20.6 ± 0.8 12.0 ± 22 288 ± 60 1775 ± 925 160 ± 28
(August24-26, 2005) Limits 3.0-16.0 40-120 18.3-27.8 6.2-31.0 132-818 482-5414 81-320
Uglich x ± SD 10.8 ± 1.4 113 ± 6 20.5 ± 0.3 102 ± 1.1 220 ± 30 620 ± 98 107 ± 14
(August22-24, 2005) Limits 4.0-19.0 100-160 19.9-23.0 5.4-15.3 98-397 367-931 29-166
Rybinsk x ± SD 11.2 ± 0.8 122 ± 14 19.9 ± 0.2 62 ± 0.6 110 ± 14 488 ± 95 55 ± 21
(August 19-20, 2005) Limits 5.5-16.5 100-170 19.2-20.5 4.1-8.4 76-171 290-919 33-92
Rybinsk x ± SD 9.4 ± 3.4 148 ± 32 21.4 ± 0.6 7.1 ± 1.6 129 ± 26 499 ± 81 124 ± 24
(August08-16, 2007) Limits 2.0-13.0 100-200 22.2-24.1 5.0-10.4 95-173 213-1162 100-182
Gorky x ± SD 8.6 ± 0.5 114 ± 4 16.6 ± 0.2 9.9 ± 0.6 203 ± 13 522 ± 88 83 ± 7
(September 02-04, 2005) Limits 6.7-13.0 90-150 15.4-17.4 4.4-13.6 89-259 173-989 38-132
Gorky x ± SD 9.3 ± 4.3 98 ± 2 28.0 ± 0.2 11.6 ± 12 306 ± 24 945±211 169 ± 32
(July 21-24, 2010) Limits 4.5-16.0 60-120 25.5-33.0 6.3-18.5 145-355 152-2295 70-349
Cheboksary x ± SD 5.1 ± 1.1 112 ± 11 17.2 ± 0.1 8.8 ± 0.4 181 ± 15 358 ± 69 109 ± 17
(September 07-08, 2005) Limits 3.0-8.0 90-140 17.0-17.4 7.7-9.8 146-220 253-489 74-144
Cheboksary x ± SD 7.2 ± 3.7 97 ± 2 27.4 ± 0.1 15.4 ± 0.9 300 ± 80 1466 ± 284 258 ± 27
(July 25-28, 2010) Limits 2.5-16.0 40-180 25.0-29.0 7.6-20.9 277-475 196-4746 119-574
Sheksna x ± SD 6.4 ± 0.9 106 ± 9 21.1 ± 0.1 62 ± 0.4 125 ± 15 332 ± 54 50 ± 5
(August03-09, 2005) Limits 4.0-13.0 60-160 20.5-21.7 3.5-8.0 59-240 104-739 29-81
Sheksna x ± SD 4.9 ± 2.0 108 ± 2 21.3 ± 0.1 7.9±0.1 142 ±32 352 ± 44 136 ± 3
(August08-12, 2007) Limits 1.3-9.0 50-180 20.5-22.7 4.9-10.8 70-188 100-744 72-260
Among the studied reservoirs, the lowest water transparency and the highest primary production of phytoplankton were recorded in the Ivan-kovo reservoir and in the Gorky and Cheboksary reservoirs in the anomalously hot summer of 2010 (Table 1). The maximum abundance, biomass and production of bacterioplankton were also recorded in the Ivankovo reservoir and in the Gorky and Cheboksary reservoirs in July 2010 (Table 1). A strong positive correlation was found between the phytoplankton primary production and NB (r = 0.68, n = 10, P = 0.05). A large number of bacteria with viruses attached to the surface of their cells were found in the reservoirs (Fig. 2). There were from 1 to 39 viruses on the surface of a cell. In July-September, the abundance of bacteria with viruses attached to their cells (NBV) varied from 12.5 (Sheksna Reservoir, 2007) to 45.0% (Cheboksary Reservoir) of their total abundance. The minimum and maximum values (NBV/NB), calculated on average for a reservoir, differed
by 1.4 times. The average NBV/NB for the reservoirs during all years of study was 23.0 ± 2.5% (Fig. 1d).
During the study period, the abundance (NPC) and biomass (BPC) of picocyanobacteria in the reservoirs varied widely from 2 x 103 to 432 x 103 cells/mL and from 0.3 to 115.0 mgC/m3, respectively. The minimum and maximum NPC and BPC, calculated on average for a reservoir, differed by 3.5 and 4.6 times, respectively (Fig. 1a, b). The abundance of picocyanobacteria with viruses attached to their cells (NPCV) varied from 8.0 (Sheksna Reservoir, 2007) to 59.0% (Gorky Reservoir, 2010) of their total abundance. The minimum and maximum values (NPCV/NPC), calculated on average for a reservoir, differed by 2.3 times (Fig. 1c). The average NPCV/NPC value for all reservoirs during all study periods was 18.2 ± 4.2%. There were from 1 to 32 phages attached to the surface of one cell.
Fig. 1. Distribution (on average for a reservoir, x ± SD) of the concentration of picocyanobacteria, proportion (% of the total abundance) of picocyanobacteria and bacteria with attached viruses, proportion (% of the total abundance) of fiee and attached viruses: a - abundance of picocyanobacteria (NPC, 103 cells/mL); b - biomass of picocyanobacteria (BPC, mgC/m3), c - proportion of picocyanobacteria with attached viruses of the total abundance of (NPCV/ NPC, %), d - proportion of bacteria with attached viruses of the total abundance of bacteria (NBV/NB, %), e - proportion (%) of free viruses (NVF/NVT) of the total abundance of virioplankton,f - proportion (%) of viruses attached to bacterial and pico-cyanobacterial cells (NVB+VPC/NVT) of the total abundance of virioplankton in reservoirs; 1 - Ivankovo (August 24-26, 2005), 2 - Uglich (August 22-24, 2005), 3 - Rybinsk (August 19-20, 2005), 4 - Rybinsk (August 08-16, 2007), 5 - Gorky (September 02-04, 2005), 6 - Gorky (July 21-24, 2010), 7- Cheboksary (September 07, 2005), 8 - Cheboksary (July 25-28, 2010), 9 - Sheksna (August 03-09, 2005), 10 - Sheksna (August 08-16, 2007)
Total abundance and structure of virioplankton. At the end of summer and in early autumn, the abundance of free viral particles (NVF) in plankton of the reservoirs varied widely; the minimum and maximum values of this parameter differed by 8.5 times (Fig. 1, 2, Table 2). The average NVF was 41.6 ± 13.4 x 106 viruses/mL for all reservoirs in the period July to early September during all years of studies. There was a positive correlation between the average NB and NVF for the reservoirs (r=0.66, n = 10, P = 0.05). A positive correlation was also found between NVF and bacterioplankton production (r = 0.63, n = 10, P = 0.05). A positive correlation was found between NPC and NVF (r = 0.52, n = 10, P = 0.05). These facts indicate that virioplankton in the reservoirs is
to a large extent represented by prokaryotic viruses. The abundance of virioplankton exceeded the abundance of bacterioplankton by 1.1-11.9 (on average 4.6 ± 1.2) times. A strong positive correlation was found between the phytoplankton primary production and NVF (r = 0.69, n = 10, P = 0.05).
The size of planktonic free viruses determined in the Sheksna Reservoir in August 2007 showed that virioplankton contained viral particles with a capsid diameter of 19 to 435 nm The average value of this parameter was 80 ± 2 nm for all water samples. The average proportions of different size groups in the total viral abundance were as follows: less than 40 nm, 10.8 ± 1.4% (limits: 0.0-23.5%), 40-60 nm, 26.4 ± 2.4% (0.0-6.8%), 60-100 nm, 39.9 ± 2.0 (34.3-52.6%), 100-150 nm, 16.5 ± 1.9% (2.6-34.2%), 150-200 nm, 4.9 ± 0.8% (0.0-10.7%) and more than 200 nm, 1.5 ± 0.5% (0.0-7.1%). Thus, viruses with a capsid diameter of 40-100 nm made the main contribution to the total abundance of free viruses. Viral particles with a capsid diameter from 26 to 254 nm were found in the virioplankton composition in the Rybinsk Reservoir in Au-gust2007. The average value of this parameter for the studied stations was 80 ± 4 nm. The average proportions of different size groups in the total viral abundance were as follows: less than 40 nm, 9.5 ± 2.6% (range 0.028.9%), 40-60 nm, 24.2 ± 5.0% (0.0-49.0%), 60-100 nm, 41.5 ± 3.7 (24.3-64.0%), 100-150 nm, 20.0 ± 5.8% (0.0-66.7%), 150-200 nm 4.2 ± 1.5% (0.0-13.6%) and more than 200 nm, 0.6 ± 0.4% (0.0-5.1%). The total abundance of virioplankton (NVT) in the Volga reservoirs varied widely, averaging 46.1 ± 14.3 x 106 viruses/mL in all reservoirs in the period between July and early September during all years of studies (Table 2). At the same time, the proportion of NVF in NVT varied within 59.9-97.6%o (on average 90.4 ± 2.6%), and the proportion of viruses attached to bacteria and picocyanobacteria in NVT varied within 3.0-40.1% (on average 9.6 ± 2.2%, Fig. 1e). The minimum and maximum values NVF/NVT and NVB+VPC /NVT calculated on average for a reservoir, differed by 1.1 and 2.1 times (Fig. 1c). The average NVF/NVT and NVB+VPC /NVT value for all reservoirs during all study periods were 90.0 ± 2.2% and 10.0 ± 2.2%. The minimum and maximum values of the ratio of the total biomass of virioplankton to biomass of their hosts (bacte-ria+picocyanobacteria, BVT/BVB+VPC) differed by 5.6 times, averaging 2.6 ± 0.8% (Table 2).
Viral infection and virus-induced mortality of heterotrophic bacteria. The frequency of visibly infected bacterial cells (FVIC) by viruses, i.e. the proportion of bacteria containing mature phage particles inside cells in NB (Fig. 2) varied from 0.5% of NB in the Sheksna Reservoir to 5.0% ofNB in the Gorky and Cheboksary reservoirs averaging 2.3 ± 0.5% ofNB (Table 3). Based on these data, it was calculated that from 3.5% to 22.9% (on average 14.8 ± 2.8%) of all bacteria in the studied reservoirs were infected by phages. Viruses infected heterotrophic bacteria of different morphology. Viruses-bacteriophage more often infected rods and vibrios (F ig. 3a).
Bacteria inside cells contained up to 860 phages/cell. The average number of phages per a water sample varied from 7 to 123 (on average 32 ± 14 phages/cell for all water samples, Table 3). Virus-induced mortality of bacterioplankton (VMB) in the Volga reservoirs varied widely (Table 3), averaging 20.7 ± 4.9% of the daily bacterioplankton production in the studied reservoirs (Table 3). In the second half of the summer, the abundance (VIMNB) and biomass (VIMBB) of bacteria that died as a result of viral lysis significantly differed both between water samples ((0.1-3.1) x 106 cells/(mL x day) and (2.2-102.4) mg C/(m3 x day), respectively) and between average values for the reservoirs (Fig. 3b, c), averaging 1.3 ± 0.8 x 10 cells/(mL x day) and 25.2 ± 16.4 mg C/(m3 x day), respectively for all reservoirs. The organic matter (VIMBB - PVB), from 2.0 to 101.6 mg C/(m3 x day), on average 21.5 ± 14.6 mg C/(m3 x day) for all water samples, was released due to viral lysis into the aquatic environment in different parts of the reservoirs. Among the studied reservoirs, the highest values (VIMBB - PVB) were recorded in the Gorky and Cheboksary reservoirs during the hot summer of 2010 (Fig. 3d). In the second half of the summer, the production of bacteriophages (PVB) varied widely in the area of the reservoirs (Table 3). The minimum and maximum values of this parameter differed by 259 times, averaging (39.5 ± 23.9) x 106 particles/(mL x day). The viral turnover time (VTT) fluctuated significantly from 0.2 to 12.8 days but the average values for the reservoirs differed to a lesser extent (Fig. 3e). VTT was on average 2.6 ± 1.3 day for all water samples.
Fig. 2. Viruses in Volga reservoirs: a - free viral particles, b - viruses attached to a bacterial cell, c - viruses inside a bacterial cell
Table 2
The abundance of free viruses (NVF), abundance of viruses attached to bacteria and picocyanobacteria, (NVB+VPC), total abundance of virioplankton (Nvr), total biomass of virioplankton (BVT), ratio of virioplankton biomass to biomass of bacteria+picocyanobacteria (BVT/BB+PC) in the reservoirs (on average for a reservoir)
NVF, 106 viruses/mL NV
, 10 viruses/mL NVT, 10 viruses/mL
BVT, mg C/m3
ByiHiiiv, %
x±SD Limits x±SD Limits x±SD Limits x±SD Limits x±SD Limits
Ivankovo (August24-26, 2005) 552 ± 9.9 15.7-120.3 5.8 ± 1.6 2.2-17.4 61.0 ± 37.0 17.9-137.7 6.1 ± 3.7 1.8-13.8 2.2 ± 0.7 1.2-3.8
Uglich (August22-24, 2005) 42.9 ± 5.3 21.7-73.8 4.1 ± 1.7 1.8-7.6 47.0 ± 18.4 23.5-81.4 4.7 ± 1.8 2.4-8.1 2.2 ± 0.6 1.5-3.1
Rybinsk (August 19-20, 2005) 30.8 ± 15.0 17.4-57.4 2.6 ± 0.8 1.8-3.8 33.4 ± 15.7 19.2-60.8 33 ± 6.1 1.9-6.1 3.0 ± 0.6 2.0-3.6
Rybinsk (August 08-16, 2007) 57.0 ± 6.5 21.1-90.5 3.4 ± 1.5 0.6-6.1 60.4 ± 24.6 25.1-93.6 6.0 ± 2.4 2.5-9.4 4.6 ± 1.4 1.8-6.6
Gorky (02-04.09.2005) 49.1 ± 18.8 21.2-85.6 4.8 ± 1.8 2.2-8.6 53.9 ± 19.4 23.6-88.8 5.4 ± 1.9 2.4-8.9 2.7 ± 0.6 1.7-3.5
Gorky (July 21-24, 2010) 47.4 ± 30.0 24.2-134.0 4.3 ± 1.8 2.2-8.6 51.7 ± 29.9 28.0-138.2 52 ± 3.0 2.8-13.8 2.6 ± 1.4 1.4-5.7
Cheboksary (September 07, 2005) 30.6 ± 2.3 26.3-36.6 4.5 ± 1.1 3.0-5.2 35.1 ± 5.0 30.8-41.8 3.5 ± 0.5 3.1-4.2 2.0 ± 0.2 1.7-2.2
Cheboksary (July25-28, 2010) 55.9 ± 10.2 38.2-77.1 73 ± 2.6 2.0-5.8 632 ± 11.2 44.8-85.9 63 ± 1.1 4.5-8.6 2.0 ± 0.6 1.3-3.4
Sheksna (August03-09, 2005) 20.4 ± 4.5 9.4-24.8 2.8 ± 0.8 1.6-3.6 23.2 ± 4.8 11.4-28.1 23 ± 0.5 1.2-2.8 2.3 ± 0.5 1.2-3.8
Sheksna (August08-12, 2007) 27.2 ± 12.8 12.4-55.7 4.0 ± 2.6 1.4-10.8 31.2 ± 13.2 19.5-65.1 3.1 ± 13 2.0-6.5 2.4 ± 1.6 1.2-6.7
a
Table 3
Frequency of visibly infected bacterial cells (FVIC), frequency of all infected bacterial cells (FIC), virus-induced bacterial mortality (VMB), number of mature phages inside bacterial cells (BS^vnal production (PV) in the reservoirs (on average for a reservoir)
Reservoirs
FVIC, % ofNB
FIC, % ofNB
VMB, % of the bacterio-plankton production
BS, phages/cell PV, 106 viruses/(mL x day)
x±SD Limits x±SD Limits x±SD Limits x±SD Limits x±SD Limits
Ivankovo (August24-26, 2005) 2.1 ± 0.8 1.2-3.6 14.0 ± 4.8 8.3-17.5 19.1 ± 83 10.5-34.8 58 ± 22 17-83 72.8 ± 39.5 11.3-131.5
Uglich (August22-24, 2005) 2.7 ± 1.4 1.4-4.9 17.0 ± 7.5 9.4-33.5 22.6 ± 10.0 10.8-40.2 45 ± 28 27-109 47.3 ± 30.2 12.2-111.4
Rybinsk (August 19-20, 2005) 2.8 ± 1.3 1.0-4.1 17.6 ± 8.0 6.9-24.3 26.0 ± 16.5 7.8-39.3 12 ± 4 7-20 12.8 ± 8.6 3.0-26.0
Rybinsk (August 08-16, 2007) 2.0 ± 0.7 1.0-3.5 13.0 ± 43 6.9-22.1 17.2 ± 7.6 7.8-34.1 22 ± 8 11-39 26.9 ± 19.4 10.4-81.1
Gorky (September 02-04, 2005) 2.3 ± 0.9 0.8-3.8 15.0 ± 5.4 5.5-23.7 20.5 ± 9.3 6.1-37.8 41 ± 18 17-72 34.4 ± 27.6 6.4-95.9
Gorky (July 21-24, 2010) 2.8 ± 1.3 1.3-5.0 17.8 ± 73 8.9-29.9 26.9 ± 15.3. 10.5-55.0 23 ± 19 7-61 74.4 ± 112.0 2.7-389.5
Cheboksary (September 07, 2005) 2.6 ± 1.1 1.8-4.0 16.8 ± 6.4 11.4-24.8 24.8 ± 12.0. 14.1-40.6 38 ± 14 24-58 39.4 ± 13.3 28.0-57.3
Cheboksary (July 25-28, 2010) 2.5 ± 0.9 0.8-5.0 16.1 ± 5.3 5.5-29.9 22.8 ± 10.4. 6.1-55.0 31 ± 35 5-123 59.3 ± 69.6 3.4-300.6
Sheksna (August03-09, 2005) 1.8 ± 0.9 0.8-3.8 11.7 ± 5.4 5.5-23.7 15.0 ± 8.7. 6.1-37.8 22 ± 11 12-47 11.3 ± 14.5 2.3-47.3
Sheksna (August08-12, 2007) 1.4 ± 0.8 0.5-3.5 92 ± 5.4 3.5-22.1 11.8 ± 8.5. 3.7-34.1 26 ± 12 8-47 22.8 ± 23.5 2.8-106.0
Fig. 3. Distribution (on average for a reservoir, x ± SD) of the proportion of bacteria of different morphology of the total abundance of lysed bacteria, abundance and biomass of lysed bacteria, the amount of organic matter released to the aquatic environment as a result of viral lysis of bacteria and turnover time of the viral abundance in the Volga reservoirs: a - proportion (%) of rods (IV), vibrios (III), cocci (II), filaments (I) in the total abundance of infected bacteria (NBFVIC) in the reservoirs,
b - abundance of bacteria lysed by viruses per day (VMNB, 106 cells/(mL x day)), c - biomass of bacteria lysed by viruses per day (VIMBB, mg C/(m3 x day)), d - amount of organic matter released to the aquatic environment as a result of viral lysis of bacteria, (VIMBB - PVB, mg C/(m3 x day)), e - turnover time of the viral abundance (VTT, day)
Viral infection and virus-induced mortality of picocyanobacteria. The frequency of visibly infected picocyanobacterial cells (FVIC) varied from 0.6% of NPC in the Sheksna Reservoir to 6.0% of NB in the Cheboksary Reservoir, averaging 2.2 ± 0.8% ofNB (Table 4). Based on these data, it was calculated that from 2.4% to 24.0% (on average 9.0 ± 3.2%) of all bacteria in the studied reservoirs were infected by viruses. Picocya-nobacteria contained up to 874 phages/cell. The average abundance of phages in the cell per water sample varied from 6 to 236 (on average 27 ± 15) phages/cell (Table 4).
Virus-induced mortality of picocyanobacteria (FMVL) varied widely (Table 4), averaging 14.2 ± 5.2% of the daily picocyanobacterial production for the studied reservoirs (Table 4, Fig. 4a). In its turn, the lysis of picocyanobacteria by cyanophages reduced the total primary production of phytoplankton on average by 1.9 ± 1.5% (range 0.4-6.0%) in the Rybinsk Reservoir, by 1.1 ± 0.9% (range 0.2-3.5%) in the Gorky Reservoir, by 1.8 ± 1.5%o (range 0.3-4.3%o) in the Sheksna Reservoir. The abundance of picocyanobacteria that died due to viral lysis (VIMPC) significantly differed both between water samples from 6.2 go 140.2 x 103 cells/(mL x day) and between average values for the reservoirs (Fig. 4b), averaging 34.9 ± 13.8 x 103 cells/(mL x day) for all reservoirs. As a result of the viral lysis of picocyanobacteria, the organic matter (VIMBPC - PVPC) from 1.6 to 23.5 mg C/(m3 x day) was released into the aquatic environment in different parts of the reservoirs, averaging 6.9 ± 2.0 mg C/(m3 x day) for all water samples (Fig. 4c). The production of picocyanophages (PVPC) also varied widely in the area of the reservoirs (Table 4). The minimum and maximum values of the production differed by 182 times, averaging (0.83 ± 0.86) x 106 particles/(mL x day). Thus, the production of picocya-nophages, on average for a reservoir, in these reservoirs was 28-219 times lower than the production of bacteriophages.
Discussion
Across the six studied reservoirs the viral abundance (VA) was the highest in the eutrophic reservoirs (15.7-134.0 x 106 viruses/mL), intermediate in meso-eutrophic reservoirs (17.4-90.5 x 106 viruses/mL) and the lowest in a mesotrophic reservoir (9.4-55.7 x 106 viruses/mL). The average virus/bacteria ratio (VBR) for a reservoir varied between 3.4 and 7.6. We found a correlation between viral and bacterial abundance (BA) and BA and VA were both positively correlated with primary production of phytoplankton. The strong positive correlation between viral and bacterial dynamics are tightly linked and support the hypothesis that the majority of virioplankton of the lakes and reservoirs are bacteriophages (Pe-duzzi & Schiemer, 2004; Ram et al., 2005; Hardbower et al., 2012). In various freshwater bodies, the abundance of free planktonic viruses enumerated by epifluorescence microscopy varied widely from 0.02 x 106 particles/mL in an oligotrophic lake (Hofer & Sommaruga, 2001) to 379 x 106 particles/mL in a hypertrophic lake (Wilhelm & Smith, 2000). In fresh waters the VBR varies from 0.03 to 41 (up to 141 in Antarctic lakes), but the VBR value is usually within 3-10 (Wommack & Colwell, 2000). In Lake Michigan, the eutrophic site was distinct with one-to two fold higher virus abundance than mesotrophic and oligotrophic sites (accordingly, 1.5-563.0 x 106, 0.5-68.9 x 106 and 0.3-29.5 x 106 particle/mL)
(Hanson et al., 2017). The authors found strong correlations between the abundances of viruses and prokaryotes and chlorophyll concentration across the trophic gradient. European mesotrophic lakes Bourget and Constance and eutrophic Lake PluBsee have appropriately high VA (34-82 x 106, 10-40 x 106 and 11-88 x 106 viruses/mL, respectively) and show clear trends in VA in relation to the trophic status (Hennes & Simon, 1995; Weinbauer & Hofle, 1998; Tomas et al., 2011). A strong positive correlation between the viral abundance and the trophic status of a water body was found for 24 Quebec lakes (Maranger & Bird, 1995).
The analysis of the literature data shows that in most freshwater bodies, the proportion of bacteria containing mature phages inside cells (FVIC) varied from 0.7% to 4.3% ofNB, i.e. from 4.9% to 26.5% of all bacteria were infected with bacteriophages, which suggests that virus-induced mortality of bacteria was from 5.4% to 45.0% of the bacteriop-lankton production (Weinbauer & Hofle, 1998; Wommack & Colwell, 2000; Simek et al., 2001; Vrede et al., 2003; Ram et al., 2005; Wilhelm & Matteson, 2008). During particular periods, the virus-induced mortality of bacterioplankton in some lakes reached 60-97% of its daily production (Hennes & Simon, 1995; Wilhelm & Matteson, 2008; Fischer & Velimi-rov, 2002).
Virus-induced mortality of autotrophic picoplankton in marine habitats varies from 1% to 21% of their daily production (Fuhrman, 1999; Ortmann et al., 2002; Mann, 2003; Baudoux et al., 2007). The studies in freshwater bodies were largely aimed at the study of the role of viruses in the control of abundance of large colonial cyanobacteria but little is known about the effect of viral lysis on mortality of natural populations of autotrophic picoplankton (Honjo et al., 2006; Tijdens et al., 2008). A close correlation between the abundances of viruses and picocyanobacteria was found in some lakes, which evidences the presence of a significant amount of cyanophages in the virioplankton composition (Colombet et al., 2009). At the same time, studies using dilution techniques and flow cytometry show that approximately 10% of picocyanobacterial loss in lakes could be attributed to viral lysis (Personnic et al., 2009).
Viruses as the most numerous component of planktonic communities in the Volga reservoirs constituted (mgC/m3) only 1.5-2.4% (on average 2.0 ± 0.3%) of the total biomass of the microbial community (viruses, bacteria, protozoa) and 0.4-1.0% (on average 0.7 ± 0.2%) of the total planktonic biomass (Table 5). The viral biomass in marine pelagic communities constitutes about 6% of the biomass of heterotrophic bacterio-plankton (Steward et al., 2007).
As a result of viral lysis of host cells (in our work, bacteria and pico-cyanobacteria), easily digestible organic compounds are released into the aquatic environment together with viruses. Thus, carbon and other nutrients in the composition of the suspended organic matter of cells were transformed into a soluble form. These soluble compounds are actively used by heterotrophic bacteria and, thus remain within the planktonic microbial community and are not channeled to higher trophic levels of food webs; it is the so-called viral shunt (Wommack & Colwell, 2000; Weinbauer, 2004). The total rate of release of organic matter of lysed bacterial and picocyanobacterial cells into the Volga reservoirs (EVIM -PV) varied from 21 to 1296 (on average 272 ± 134) mg C/(m2 x day) or from 6.8 to 132.2 (on average 40.7 ± 16.8) mg C/(m3 x day) (Table 6). The lysed bacteria constituted the main traction in EVIM - PV (Fig. 4d).
The viral shunt was an important source of the easily digestible organic matter input: the EVIM - PV values were 6.3-52.9% (on average 18.4 ± 5.2%) of the phytoplankton primary production for m2 and could provide 3.4-31.6% (on average 7.0 ± 1.4%) daily organic carbon demand for heterotrophic bacterioplankton (Table 6). Fischer & Velimirov (2002) estimated that in a eutrophic lake, on average 15.2 ^g C/(L x day) was released into the aquatic environment due to viral lysis of bacterial cells, which was 46% of the daily bacterial production in the water column or about 14% of daily demands of bacteria for organic matter.
The abundance of planktonic viruses in natural waters depends both on their production and decay or extraction of viral particles from the water column (Bratbak et al., 1994). The abundance of viruses in the water column may decrease due to the following reasons: grazing of heterotrophic nanoflagellates (HNF) on free viruses (Suttle & Chen, 1992; Gonzalez & Suttle, 1993; Bettarel et al., 2005), sedimentation of large suspended particles with attached viruses; decay by bioactive molecules
such as exoenzymes, proteases, or nucleases, which extract nucleic acids from viral capsids (Bratbak et al., 1994; Noble & Furman, 1997, 1999), mortality caused by viriophages (La Scola et al., 2008; Fischer & Suttle, 2011; Yau et al., 2011). The experimental studies have demonstrated that the grazing rate of HNFs on free viruses is low, from 1.9 to 3.3 virus-es/(ind. x h) (Suttle & Chen, 1992; Bettarel et al., 2005). HNF can, apparently, extract only large viral particles from water.
During the period of study, the abundance of free viruses with a cap-sid diameter more than 0.2 pm (the size of the smallest bacteria about 0.2 pm) varied from 0 to 2.2 x 106 viruses/mL in the Rybinsk and Sheksna reservoirs. Using the clearance rates we found that in the reservoirs, HNF grazing rates on large viral particles could be 1. 1-12.0 (on average 5.0 ± 3.8 virusesfagellate x h)), i.e. the number of free viruses grazed by the HNF community was low (Table 7).
Fig. 4. Distribution (on average for a reservoir, x ± SD) of the production of picocyanobacteria, abundance of lysed picocyanobacteria, amount of organic matter released to the aquatic environment as a result of viral lysis of picocyanobacteria and proportion of heterotrophic bacteria and autotrophic picocyanobacteria of the total amount of organic matter released to the aquatic environment as a result of viral lysis in the Volga reservoirs:
a - picocyanobacterial production (PPC, mg C/(m3 x day)), b - abundance of picocyanobacteria lysed by viruses per day (VIMNPC, 103 cells /
(mL x day)), c - amount of organic matter released to the aquatic environment as a result of viral lysis of picocyanobacteria, (VIMBPC-PVPC,
mg C/(m3 x day)), d - proportion of heterotrophic bacteria (HB) and picocyanobacteria (PC) in the total amount of organic matter released into aquatic environment as a result of viral lysis of heterotrophic bacteria and picocyanobacteria (EVIM-PV, mg C/(m3 x day)) in reservoirs: 4 - Rybinsk (August 08-16, 2007), 6- Gorky (July 21-24, 2010), 8 - Cheboksary (July 25-28, 2010); 10 - Sheksna August 08-16, 2007)
Table 4
Frequency of visibly infected picocyanobacterial cells (FVIC), frequency of all infected picocyanobacteiial cells (FIC), virus-induced mortality of bacteria (FMVL), number of mature phages inside picocyanobacterial cells (BS), virus-picocyanobacteiia production (PVPC) in the reservoirs (on average for a reservoir)
Reservoirs FVIC, % NPC FIC, % Npc FMVL, % ofpicocya- bs j. nobacterial production ages/cell PVPC, 106 viruses^mL x day)
x ± SD Limits x ± SD Limits x ± SD Limits x± SD Limits x ± SD Limits
Rybinsk (August08-16, 2007) 2.6 ± 1.4 1.0-5.3 10.4 ± 6.8 4.0-21.2 17.2 ± 15.2 7.6-52.0 21 ± 16 6-58 0.72 ± 0.56 0.11-1.62
Gorky (July 21-24, 2010) 1.8 ± 0.8 1.0-3.4 7.2 ± 3.3 4.0-13.6 11.2 ± 5.7. 6.0-22.7 13 ± 4 8-21 0.34 ± 0.23 0.06-0.83
Cheboksary (July 25-28, 2010) 3.2 ± 1.5 0.8-6.0 12.8 ± 5.8 3.2-24.0 19.7 ± 10.4. 4.8-45.6 48 ± 68 6-236 2.08 ± 3.52 0.07-10.95
Sheksna (August08-12, 2007) 1.4 ± 0.8 0.6-2.9 5.6 ± 4.6 2.4-11.6 8.6 ± 5.5 4.5-24.5 25 ± 29 5-81 0.19 ± 0.15 0.06-0.45
Table 5
Proportion (%) of different components of plankton in total biomass of planktonic community (TBP, mg C/m3) in the Upper and Middle Volga reservoirs
Reservoir Total biomass % of TBP
of plankton viruses phytoplankton bacteria Protozoa metazooplankton
Ivankovo (August24-26, 2005) Uglich (August22-24, 2005) Rybinsk (August 19-20, 2005) Sheksna (August03-09, 2005) Gorky (September 02-04, 2005) Cheboksary (September 07, 2005) Cheboksary (July 25-28, 2010) 871 500 567 603 711 627 1531 0.7 1.0 0.6 0.4 0.8 0.6 0.4 373 29.8 59.7 54.7 61.6 68.8 63.0 32.1 40.1 20.6 20.3 29.4 26.8 25.0 6.9 7.6 5.7 6.1 3.5 1.0 3.6 23.0 21.5 13.4 18.5 4.7 2.8 8.0
Table 6
The amount of organic matter released to aquatic environment due to viral lysis of bacteria and picocyanobacteria, (VM-PV), primary production of phytoplankton per m2 (aPPHY), daily demand of bacterioplankton for organic matter (CB) in Volga reservoirs
Rybinsk, 2007_Gorky, 2010_Cheboksary, 2010_Sheksna, 2007
Parameters x ± SD Limits x± SD limits x ± SD Limits x ± SD Limits
LVIM-Pv, mg C/(m2 x day) 208±103 52-418 384±374 28-1039 384±295 83-1296 114 ± 75 21-301
ZPphy, mg C/(m2 x day) 1202 ± 775 517-2593 1485±951 246-2922 2776±1960 740-8395 687±350 242-1141
LVIM - Pv/SPpHY, % 17.3 ± 10.7 6.3-37.7 25.8 ± 16.5 1.5-52.9 13.8 ± 16.4 3.3-63.4 16.6 ± 10.2 2.7-47.5
VIM - Pv, mg C/(m3 x day) 29.0 ± 10.1 12.9-44.0 49.8 ± 42.6 9.9-132.2 59.7 ± 35.7 16.5-125.1 24.4 ± 10.6 6.8-46.1
CB mg C/(m3 x day) 413 ± 81 333-607 564±350 232-1164 860±396 397-1913 453±163 240-867
VIM - Pv/ CB. % 7.0 ± 2.6 3.4-12.1 8.8 ± 42 4.3-16.3 6.9 ± 3.3 2.7-17.4 5.4 ± 6.6 1.4-31.6
Table 7
Grazing rates on free viruses of 0.2 ^m and more (CVF), grazing rates on bacteria (CB), grazing rates on viruses attached to bacterial cells (CVB) and grazing rates on viruses inside bacterial cells (CBS) by natural populations of heterotrophic nanofagellates in Volga reservoirs
Parameters
Rybinsk Reservoir
Sheksna Reservoir
x± SD
limits
x ± SD
limits
CVF, 106 viruses/(mL x day) 0.04 ± 0.12 0.00-0.43 0.15 ± 0.04 0.04-0.46
CB, 106 cells/(mL x day) 22 ± 12 0.9-4.7 1.7 ± 0.7 1.0-2.8
CVB, 106 viruses/(mL x day) 12 ± 0.6 0.1-2.4 0.9 ± 0.8 0.2-2.7
CBS, 106 viruses/mL x day) 6.1 ± 3.8 2.1-12.0 4.4 ± 3.8 0.7-13.9
CVF + CVB+ CBS, 106 viruses/mL x day) 73 ± 42 2.2-14.8 5.45 ± 43 0.9-17.0
Cvf + Cvb+ Cbs / Pv, % 30.6 ± 15.4 13.1-59.8 27.6 ± 13.7 10.0-52.8
A much greater number of viruses reach a higher trophic level when HNF graze on infected bacteria and bacteria with viral particles attached to their cells (Table 7). As a result, a significant amount of viral particles were involved in the food web of the reservoirs, which constitutes a significant percentage of the daily virioplankton production (Table 7). However, the biomass of grazed viruses in the Rybinsk and Sheksna reservoirs (0.73 ± 0.42 mg C/(m3 x day) and 0.54 ± 0.43 mg C/(m3 x day), respectively) was 53-58 times lower than the biomass of grazed bacteria (38.8 ± 5.2 mg C/(m3 x day) and 31.3 ± 2.6 mg C/(m3 x day), respectively).
Conclusions
The results of the study on ecology of planktonic viruses indicate that virioplankton is an important structural and functional component of the microbial planktonic food web in the Upper and Middle Volga reservoirs. Viruses cause the mortality of a large number of autotrophic and heterotrophic picoplankton. Due to the viral lysis of heterotrophic bacteria and picocyanobacteria cells, a significant portion of their production does not enter a higher trophic level and products of the viral lysis increase the reserves of soluble and insoluble organic matter, i.e. they are retained within the microbial communities. Thus, viruses support their hosts providing them with organic carbon and minerals. Viral particles are involved
in the food webs of water bodies when they are directly grazed by protozoa or with host cells. The quantitative analysis of these processes in the studied reservoirs indicates a substantial role of planktonic viruses in carbon fluxes through the planktonic food webs.
This research was financed by 1he Ministry of Science and Higher Education of the Russian Federation and was carried out within Hie framework of a state assignment no. 121051100102-2.
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