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Distribution of marine viruses in the Central
and South Adriatic Sea

ARTICLE in MEDITERRANEAN MARINE SCIENCE · SEPTEMBER 2014
Impact Factor: 1.73 · DOI: 10.12681/mms.911

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Marin Ordulj Nada Krstulovic
University of Split Institute of Oceanography and Fisheries
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2003. (Fuhrman. JOZIĆ2 and M. 2012). Jacquet et al. 2010). which coincided with lower HNF abundance and vice versa during the warmer part of the year. Fuhrman. Adriatic Sea. al.. coastal area of the central and southern Adriatic Sea. Suttle. Research Article Mediterranean Marine Science Indexed in WoS (Web of Science.. KRSTULOVIĆ2.hr Handling Editor: Lydia Ignatiades Received: 16 May 2014. 65-72 65 . Viral abundance ranged from 3. Weinbauer.. In a comprehensive study. In the Adriatic Sea. Suttle. 2005).medit-mar-sc. ŠOLIĆ2 1 University of Split. Introduction the virally induced mortality of bacteria in the marine en- vironment can affect the flux of nutrients and organic mat- Viruses are the most abundant and ubiquitous compo- ter by increasing their recycling through the microbial loop nent of marine microbial plankton (Thingstad et al. The ratio between viruses and bacteria (VBR) was higher during the colder part of the year. Distribution of viruses. 1994. a 10–20% loss of bacterial production (Heldal & Bratbak. which can affect bac. 1999). as a major predator of bacteria. 1991. bauer et al. 2000. Karuza et al. 1994. Despite their small es were carried out mostly in the northern Adriatic Sea (Wein- size. tats worldwide. S. complete distribution data for marine vi- ied in the last two decades. 2015. 1999. This suggests that HNF grazing could be more important in controlling bacterial abundance during the warmer part of the year.911 Distribution of marine viruses in the Central and South Adriatic Sea M.. 1993. they represent a substantial biomass in the marine en. and viral lysis during the colder part of the year. and was on average 25-fold higher than bacterial abundances at all investigated stations. 2004. Weinbauer & Peduzzi. ISI Thomson) and SCOPUS The journal is available on line at http://www. 2005. University Department of Marine Science. Corinaldesi et al. Fuhrman. the microbial loop has been recognized and heavily stud.. it is generally accepted that viruses are responsible rine bacteria and heterotrophic nanoflagelates in the coastal for about 10–70% of total bacterial mortality and can cause and open Adriatic Sea. Wommack & Colwell.55 to 27.32 × 106 virus-like particles mL-1. The abundance of viral. mined the viral abundance throughout the Adriatic Sea. 2005). al. 1999. the percentage of high (HNA) and low (LNA) nucleic acid bacteria in the total number of bacteria and the distribution of heterotrophic nanoflagellates (HNF). Viral counts typically range from 104 to 108 whereas data from the eastern coastal Adriatic Sea are scarce.. Marine viruses are significant ruses along the eastern coast of the central and southern Adri- agents in controlling the marine bacteria and phytoplankton. especially in the area influenced by the rivers Krka and Jadro. D. Bratbak et al. The highest viral abundances were found at coastal stations. N. Croatia Corresponding author: marin.net DOI: http://dx. Livanjska 5/III. The important role of viruses in did not collect data from Croatian territorial waters. Studies of marine viral abun. atic Sea and in the open central Adriatic are presented. Sci. 21000 Split. Stopar et dance have been performed in various locations and habi.org/10. viruses cause the release of particulated (POC) and dissolved (DOC) organic carbon. Bacteria. Keywords: Viruses. 1995. Heterotrophic nanoflagellates. ORDULJ1. Additionally. 2003. Luna et al. (2003) deter- Viruses are in general an order of magnitude more abun. 2008). 21000 Split. Accepted: 15 September 2014. 1993. The and represent a major force behind biogeochemical cycles main purpose of the study was to demonstrate the seasonal (Fuhrman. In this paper. By lysing ma- Materials and Μethods rine bacteria. 2010. POB 500. 2002. Wommack & Colwell. and Medit.. were studied as well. per mL-1 depending on the trophic state of the study area.ordulj@unist. nanoflagellates was determined at 23 stations across the terial carbon cycle (Bongiorni et al. ŠANTIĆ2. whereas the lowest values were found in the open sea and in the coastal area of the southern Adriatic. Published on line: 28 November 2014 Abstract The seasonal distribution of marine viruses and their relationship with heterotrophic bacteria in the coastal and offshore area of the central and southern Adriatic was studied.doi.. Corinaldesi et al. 2007).. 1999.12681/mms. distribution of marine viruses and their relationship with ma- Today. Weinbauer et vironment (Kirchman. Mar. but dant than marine bacteria. the studies of marine viral abundanc- 2000. 16/1.. Croatia 2 Institute of Oceanography and Fisheries.. Furthermore. bacterial and heterotrophic terial community structure and have an impact on the bac. Bongiorni et al.. No significant difference was established in the relationship of the viruses with HNA and LNA bacterial groups.

and 3H is the integrated 3H-thymidine incor- were incubated in the dark for 20 min and mounted on poration rate during the experiment.02-µm filters N1)/3H. The abundance of marine viruses was determined as Bacterial cell production was determined using the previously described in Noble and Fuhrman (1998).5×). Al2O3. CA011). 1980). Fig. 1987) CF=(N2- samples (2 mL) were filtered through 0. content (Jochem.250× (objective 100×. scope slides were observed with an Olympus microscope The abundance of marine bacteria was determined under UV light illumination at a magnification of 1000× by flow cytometry (Beckman Coulter Epics XL MCL) (Porter & Feig. final concentration) and frozen at tion were calculated from bacterial cell number and 3H- -80°C until analysis. light scattering. 2001).at two stations located in the open sea area of the central 1μm yellow-green beads were added (Level-III Epics Adriatic (Fig.. 1 mL samples after testing the normal distribution of the data.5% ascorbic acid. Two September 2010 to September 2011. and SeaBird 25 CTD profiler. Mar. b) Coastal areas: Kaštela Bay (ST101–ST104). PG2). containing 0. Samples for hetero. with 3 H-Thymidine incorporation techniques (Fuhrman & slight adjustments.8-μm pore di- nification of 1. open sea stations (CA009. The number of heterotrophic nanoflagellates (HNF) pH 7. diameter: 25 mm. c) Coastal areas: Ploče (P1–P3). Ireland). glass slides with a drop of 50% phosphate buffer (6. 1). Whatman) and stained bacteria at the beginning and the end of the experiment. and ameter polycarbonate filters (Millipore. as described in Marie et al. Temperature and salinity were recorded using the acid (HNA) and the low nucleic acid bacteria (LNA). Viral counts was estimated using epifluorescence microscopy. ocular 12. Filters respectively. Zadar (Z1–Z3). 2015.2 μl s-1). 65-72 . 16/1. groups of bacteria were distinguished according to their lected using 5-L Niskin bottles at standard oceanographic relative green fluorescence as a proxy for nucleic acid depths from the surface to the seabed in 5 to 10 m inter. the water column and between investigated stations. Preserved pre-filtered seawater (Riemann et al. Du- brovnik (D1–D3).8) and 50% glycerol. 1: Study area with sampling stations: a) Coastal areas: Pag (PG1. Sam- were obtained by epifluorescence microscopy (Olympus ples were stained with 4-6-diamidino-2-phenylindole BX 51. The relationship among the investigated parameters trophic bacteria were preserved in 2% formaldehyde and was determined using Pearson’s rank correlation index stored at 4 °C until analysis (5-10 days). CA007). Šibenik (Š1–Š3). 66 Medit. Collected samples were preserved Azam. Analysis were stained with SybrGreen I and analysed on the Beck. Conversion factors for bacterial produc- in formaldehyde (2%.7 mM. Split (S1–S5. which was performed in the labora. To standardise fluorescence intensity of cells. Slides were stored at -20°C until analysis.. (1997). (DAPI) for 10 min and filtered through 0. with SYBR Green I (stock solution diluted 300×). Sampling was carried out monthly from Division of Coulter Corporation Hialeah. Micro- are expressed as virus-like particles (VLP) per mL. equipped with a blue excitation filter) under mag. where N1 and N2 represent the numbers of (Anodisc. of variance (ANOVA) and t tests were used to determine man Coulter EPICS XL-MCL (high flow rate from 1 to the differences in microbiological parameters throughout 1. Florida). thymidine incorporation during bacterial growth in 1 μm tory within 24 hours after the end of the cruise. Samples were col. 1980). Sci. referred to as the high nucleic vals.

numbers between surface and bottom layers were deter- tive coastal sea to the open sea area along the trophic gradi. with a mean of southern Adriatic coastal sea area. abundance for the oligotrophic part of the eastern Mediter- Notably..24 ± 3. Previous studies of various environmental a mean of 7. Adriatic was less pronounced compared to that in the central 2012).26 × 106 VLP mL-1. viral abun. (1993) also determined a decrease in viral number following a transect Viral abundance and distribution from the river Po to Rovinj in the north Adriatic. p < 0. 2003. Magiopoulos et al. Results obtained parameters (inorganic salts. Bongiorni et al. which dance was similar to that in the southern Adriatic coastal sea agrees with our data for the open central and southern area and ranged from 3. 2: Abundance of viruses along the trophic gradient following Fig. sea stations from Pag to Cavtat. with a mean of 7. where viral abundance reached 27.. 2003. virus-to-bacteria ratio (VBR).74 38. 2).51 Sea Medit. phic nanoflagellates. P < 0.85 × 106 VLP mL-1.89 11. who observed a decrease in viral tribution of marine viruses in the coastal and offshore area number following a transect from the river Po to the open of the central and southern Adriatic Sea.51 ± 1.45 0. with a 2. in the sampling period fall within the range of previously heterotrophic bacteria.12 0. bacterial production and HNF) in published data for the coastal and estuarine environments of the coastal area of the south Adriatic Sea also showed that the north Adriatic Sea (Weinbauer et al.01. The lowest viral abundances 1 .93 to 11.04 37. 16/1.50 ± 0. heterotrophic bacteria.33 57.24 × 106 VLP mL-1. with a mean of 11.20 × 106 VLP mL-1 in the surface layer and in the Šibenik area in the surface layer of station Š1.18 ± 0. 2003.. 2015. Significant differences (P < 0.31 ± 0. percent- age of LNA and HNA bacterial groups and abundance of HNF at the investigated coastal and open sea stations (mean value ± SD).03) stations. bacterial abundance.66 ± 5. 1995.14 × 106 VLP mL-1 with Adriatic Sea.63 ± 0. Cori.71 0. 3: Mean annual values of viral abundance at the sampling the Kaštela bay to Palagruža Island transect (mean value ± SD).5-fold higher abundance in the eutrophic dance of marine viruses. the mean values of bacterial produc.25 7.10 42.Results and Discussion (ANOVA. (t-test. 2001.32 × 106 VLP mL.21 24.32 × 106 VLP mL-1. ANOVA) in viral tion and the virus-to-bacteria ratio (VBR).24 × 106 VLP mL-1. naldesi et al. Magagnini et ers at all investigated coastal (t-test.76 ± 6. Table 1. as well as a difference in Viral abundance in the mid-Adriatic coastal sea area viral numbers along a trophic gradient from the coastal to ranged from 4.. 65-72 67 . The mean abun. Stopar et al.51 ± 1.49 ± 2. These stations Fig.63 ± 8.71 ± 3. 1993. salinity. Sci. similar to This study presents the first record of the seasonal dis.05) and open sea al. Mar. bacterial production (BP)..49 ± 2..97 37. abundance in oligotrophic waters is 6 × 106 mL-1.94 ± 1. stations (mean ± SD). mined at station ST101 in Kaštela Bay with a maximum ent. temperature abundance were observed along the investigated coastal and salinity in the investigated area are shown in Table 1. 2007. Temperature. sea area. following the Kaštela bay to Palagruža Island transect value of 20.34 ± 5. viral abundance.. 2004.43 ± 0.. p < 0..01 62. 2010). Boras et al. from 3.84 × 106 VLP mL-1 to 27.59 ± 9.. Significant differences were observed in the vertical 2005.. Tempera- Salinity Viruses Bacteria BP HNA LNA HNF ture VBR (‰) (106 mL-1) (106 mL-1) (104 mL-1 h-1) (%) (%) (103 mL-1) (°C) Coastal 16. (2005). 3). the open sea area (Fig. heterotro. Hwang & Cho (2002) stated that the typical viral Adriatic.08 Sea Open 17. 2009 Magiopoulos et al. In the central Adriatic open sea area.55 to 13.. 2012). biomass of phytoplankton.01) (Fig. 2012) and the Mediterranean Sea distribution of viruses between the surface and bottom lay- (Alonso et al. Bongiorni et al. than in the oligotrophic area. which is similar to the viral VLP mL-1. Weinbauer et al. Karuza et al.53 ± 3. Weinbauer et al.85 0.56 ± 3.31 ± 1. 2010..33 0. The extreme variations in viral The viral abundance decreased from the more produc.57 × 106 7.08 24. the fluctuation in viral abundance in the southern ranean Sea (Weinbauer et al. this area can be classified as oligotrophic (Šantić.37 ± 8. and in the were determined in the Dubrovnik area.01 0.11 ± 0.

49. n = 130. whose various environmental parameters or lysogeny could be waters are rich in organic matter and nutrients. p < during the summer and late autumn period (Weinbauer 0. whereas a low.. the most common reason but it was discarded in a paral- sponsible for a higher abundance of heterotrophic bacteria. viral replication and are an important factor in the control minate the cause. ported by Jacquet et al. a high viral abundance was observed investigated coastal sea stations (r = 0. this might result in an increased viral abundance in assumed that HNF significantly controlled the bacterial the surface layers of estuarine areas (Weinbauer et al. might be a substantial increase in the abundance of het- ously published data. The determined many potential causes behind the relative low viral num. summer and at the end of autumn. and ultimately probably for sible explanation for this unexpected summer minimum the higher abundance of marine viruses. 4: Mean monthly abundance of viruses (a). heterotrophic bacteria (b).05) (Table 2). p < 0. The summer HNF abundance was three- which can be related to an input of viral particles with riv. population during the warmer part of year. Sci. Similar results was re. than the abundance during the winter. lel study we conducted (unpublished data). 2015. Although there are (r = 0. tions (Fig. fold and two-fold higher at coastal and open sea stations. In the between marine viruses and heterotrophic bacteria at the northern Adriatic. We observed that viral abundance started during spring and autumn at the investigated coastal sta. 4c). whereas vi- Maranger & Bird. This is re.. n = 490. We layer. 1995). 68 Medit. HNF (c) and mean monthly bacterial production (d) and virus/bacterium ratio (e). correlations suggest that bacteria were the main host for bers found in early summer. 65-72 . erine water. 1995).58.. We determined statistically significant correlations er viral abundance was observed during the winter. Since fresh water spreads on top of the surface respectively. ruses controlled the bacterial population during the cold- The present study showed the highest viral abundance er part of year. to increase with a decrease in the abundance of HNF and tions in the central and southern Adriatic Sea. which represents viral abundances were observed in early summer (June) the beginning of viral domination over bacterial popula- at all sampling stations (Fig.05) and at the open sea stations of the central Adriatic et al.. we were not able to deter. 16/1. Decay of viral particles in response to of viral abundance.are heavily influenced by the Jadro and Krka rivers. pling stations. A similar correlation was found by Fig. According to previ. The lowest bacteria in the investigated coastal area. 1993. Another pos- which are the main viral hosts. Weinbauer et al. (2010) who stated that a higher abundance of marine viruses was observed during early Viruses in relation to heterotrophic bacteria spring. 4a). 1993. Mar. a negative correlation between viral erotrophic nanoflagellates at coastal and open sea sam- abundance and salinity was observed in the estuarine area.

59 0. the previously mentioned sampling stations.32 -0.68 0.31 -0.25 n. A low correlation between marine viruses and hetero- Drniš and Knin.20 -1.40 1.05 Viruses 1. there is no dependence of marine viruses on any bacterial which enriches this area with nutrients that stimulate a high group regarding their nucleic acid content.43 0.33 n.63 0. the HNA bacterial group at coastal sea and the LNA bac- The sampling station situated in the Vranjic Basin terial group at open sea stations.05).00 -1.2 0. which is stations. -0.56 HNA 0. n.0 1.26 -0. heterotrophic nanoflagellates (HNF).s. whereas at open sea bacterioplankton (Šantić et al.2 0.00 0.51 -0.57 -0.2 0. < 0. P<0.5 n.s. -0.32 0.s. 2015.00 0.74 1.58 1.27 0. n.54 n. This demonstrates that (Kaštela Bay) is under the strong influence of River Jadro. -0.07 1.0 -0.s. At all stations.s.s. (1993) in the Mexi. n.57 0.0 1. 0.18 -0.49 n.Weinbauer et al.00 Medit.24 -0.74 0.37 0.54 n.60 HB 0. (2003) in the abundance of both bacteria and viruses (Šolić et al.74. heterotrophic bacteria (HB).4 0.17 1.34 0. n..32 0.s.00 Š1 and ST103 Viruses HB BP HNF HNA LNA Temperature Salinity n=49.s.31 n. n.s.05 Viruses 1.22 -0. n.34 n.40 -0.2 -0.25 -0.54 LNA -0. 2012) and eventually virio.2 0. salinity and viral abundance at coastal sampling stations cal correlation between marine viruses and heterotrophic was negative and lower (r = -0.20 0. -0.0 -1. 0.s.00 0.00 n. 1.49 1.40 0. n. 2010).34 n.25 -0.s.24 -0. 0.22 -0. -0.44 -0. 16/1.05) than bacteria was even stronger (r = 0.33 -0.00 n.34 n.25 LNA 0. which is reflected by low salinity in the trophic bacteria groups with a high (HNA) or low (LNA) surface marine layer (Šantić. n.s.24 -0. Pearson’s correlation coefficients between viruses.45 -0. n.00 0.18 1.43 n.59 0.01 -0.s. (1993) and Stopar et al.05). HNF -0.24 0. Results in this paper also demonstrate a high negative sta- can Gulf and by Cochlan et al.32 HNF 0.24 0.00 n.31 0.s.00 0. n. bacterial production (BP).00 0.34 Temperature -0.s.01 0. 1..05 Viruses 1.37 -0.s.31 1. 1. Salinity -0. 2010). the statisti.s.26 0.43 0. p < 0.47 1.00 0.24 0.0 n. Čikola and Gudaća Rivers and is also under Viruses in relation to HNA and LNA bacterial groups the influence of the waste waters of the cities of Šibenik. 65-72 69 .s.18 -1..34 n. -0. n = 49.3 0. 0.40 -0.68 1.24 0.17 -0.63 0.54 BP n.17 0. 2010).00 0. north Adriatic Sea.s.25 -0.61.25 0.00 0.. n. 0.34 -0. P<0.54 0. n. 1.s.40 BP 0. Salinity -0.5 0.49 -0. 0.61 HB 0. n. -0.58 0. The statistical correlation between area and Kaštela Bay were analysed separately.00 n.s.00 0.45.s. that at the open sea sampling stations (r = -0.45 HB 0. p At the same sampling stations. a high nucleic acid content was determined at all investigated concentration of organic and inorganic matter. Mar.s. where lower When the results from sampling stations in the Šibenik salinity was measured.56 -0. This plankton since viral abundance is closely correlated with might demonstrate the dependence of marine viruses on the abundance of bacteria (Jacquet et al.0 -1. Coastal Sea Viruses HB BP HNF HNA LNA Temperature Salinity n=490.00 Open Sea Viruses HB BP HNF HNA LNA Temperature Salinity n=130. was also found.s.47 0.0 n.61 -0. n. a significant statistical cor.s.3 -0.2 -0. (1993) in the southern part tistical correlation between salinity and viral abundance at of the Californian Bay and the Chucki Sea.0 0.3 0.4 -0.49 0.00 n.s.3 -0. stations they were negatively correlated (Table 2). -1. p < 0. The area near Šibenik is greatly influenced by the Krka.18 0.07 Salinity -0.34 LNA -0.25 Temperature n.60 -0.s. n = 490. 0.20 HNA -0.27 0. Table 2. Data for the correlation between viruses and other relation between bacterial production and viral abundance investigated parameters are shown in Table 2.2 -0.2 1.3 -0.s.s. 1.24 -0. Marine viruses and HNA bacteria at coastal sea transported by the riverine water stimulates the growth of stations were positively correlated.s.25 HNF 0.40 HNA 0.s.54 Temperature 0.3 0.17 0.44 n.s. Sci. n = 130.20 1.s.s. by Boehme et al. temperature and salinity.00 1.s. P<0. 1.43 n.s. low nucleic acid (LNA) and high nucleic acid (HNA) bacterial groups.s.31 n.51 BP 0.31 0.00 -0.s.00 0. Accordingly.

which is mean annual values of LNA and the HNA heterotrophic bac- responsible for the better survival of this bacterial group. p < 0. whereas at all other stations.LNA bacteria slightly dominated over HNA bacteria. p < 0. 2015. whereas the VBR at the open sea sta. Jochem et al. 5: Mean annual value of viral abundance compared to an oligotrophic environment than HNA bacteria. n = 130. southern and central Adriatic Sea varied from 8 to 76. 7). The prevalence of LNA bacteria can be explained by their morphological characteristics and better competi- tion for food resources in an oligotrophic environment. Moreover. 7: Mean annual values of virus to bacteria ratio at the sam- The VBR determined for the coastal stations of the pling stations (mean ± SD). The abun- similar to the values determined for the northern Adriatic dance of HNF was high during the warmer part of the year Sea. r = -0. This greater abundance of HNA bacteria is related to a higher trophic state and to higher bacterial production in the area.. At the open sea stations. The VBR at the open sea stations of the central Adri. since LNA bacteria have smaller cells and a higher surface-to-volume ratio.4.1 LNA. when bacterial abundance ern part of the central Adriatic. 2010). The highest VBR was determined in the north. at the coastal sea stations. 2004). The VBR in different marine environ- ments varies from 3 to 100 with a mean of 25.. The abundance of LNA bacteria was slightly greater than that of HNA bacteria in the open sea area of the cen- tral Adriatic (Fig. 1998. to the winter VBR. we observed a strong domi- tions of the central Adriatic Sea varied from 14 to 40.05) in this area. when a high abundance of bac- nance of viruses over bacteria within this area (Fig. terial predators (HNF) was observed (Fig. 5). 65-72 .. was determined during March. which influences viral produc- tion and thus increases the VBR (Jacquet et al. a mean of 25 ± 6. Sci.34. A higher VBR is characteristic of eutrophic and more productive sea areas. 16/1. The VBR for coastal sea stations was when lower abundances of HNF were recorded. suggesting that viruses generally did not Fig. n = 49. A signifi- cant statistical correlation between viruses and HNA bacte- ria was determined (r = 0. Virus-to-bacterium ratio The virus-to-bacterium ratio (VBR) is a good parameter that can be used to monitor the predominance of viral abun- dance over heterotrophic bacteria in the marine environment (Jacquet et al. coastal (a) and open (b) sea areas. 6: Mean monthly viral abundance in relation to the HNA show preference to any particular bacterial group accord. followed by high viral and bacterial abundance. a lower VBR ranean Sea. teria in the study area. (2001) observed that they have a higher specific growth rate in Fig. and LNA bacterial groups during the sampling period for ing to their nucleic acid content. Fig.05). n = 441. causing a lower VBR. which is the opposite to that in the other investigated areas.1 HNA. p < 0. with nance of viruses over bacteria in February. In the coastal sea area. Exceptions to our results were observed at stations in the Šibenik area (Š1) and Kaštela Bay (ST103). in most cases. and during July.. which has been previously described for the southern and central Adriatic Sea by Šantić (2012) (Fig. compared atic was within the range of data reported for the Mediter. Therefore. 2010). viral abundance showed better correlation with the most abundant bacterial group (HNA or LNA). May and June. Zubkov et al. Nutrient-rich environments stimulate higher growth and production of bacteria. with a mean of 25 ± 10. was high. suggesting a high domi. where a slight predominance of HNA bacteria was determined. Mar. 6) and statistical correlation showed that marine viruses were more dependent on this group of bac- teria than on HNA bacteria (r = 0. 70 Medit. compared to HNA bacteria (Button.05). the correlation between viruses and HNA or LNA bacteria was low as mentioned previously (r = 0. 4e).

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