AQUATIC MICROBIAL ECOLOGY Vol. 48: 13–18, 2007 Published June 20 Aquat Microb Ecol Solar radiation-driven decay of cyanophage infectivity, and photoreactivation of the cyanophage by host cyanobacteria Kai Cheng1, 2, 3, Yijun Zhao2, Xiuli Du2, Yaran Zhang2, Shubin Lan2, Zhengli Shi1,* 1State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, PR China 2Hubei Key Laboratory of Urban Water, Environmental Ecology, Central China Normal University, Wuhan 430079, PR China 3Graduate School of Chinese Academy of Sciences, Beijing 100039, PR China ABSTRACT: Cyanophage PP (isolated from Plectonema boryanum and Phormidium foveolarum and named after the respective first letters of its 2 hosts) is a short-tailed, icosahedral-shaped, and double- stranded DNA virus and can be frequently detected with a high abundance and activity in many eutrophic lakes in Wuhan City, PR China. To understand how the virus survives solar UV-B damage, we examined the decay of cyanophage PP induced by solar UV-B radiation and the photoreactivation repair by host cyanobacteria, on 1 d in each of the 4 different seasons throughout a year. The UV-B transparent or non-transparent bag containing the cyanophage PP was exposed to sunlight at differ- ent water depths and was collected at varying time points. The collected sample was treated with or without radiation of a photoreactivating wavelength (300 to 500 nm). The decay ratio (here desig- nated as the percentage of infectivity lost) for cyanophage PP from all samples caused by UV-B radi- ation ranged from 29.8 to 92.1%. Season and water depth were the main factors influencing the decay ratio, while long exposure time had less effect. The repair ratio (here designated as the per- centage of revived infectivity by photoreactivation) for cyanophage PP reached a maximum during the first 1 to 3 h of exposure, at all depths. The maximum repair ratios of photoreactivation for each of the decayed samples, ranging from 7 to 59%, were negatively correlated with the decay ratio. In most cases, the repair ratio of photoreactivation reached a maximum in the first 4 h. Our results indicate that solar UV-B radiation may decrease the infectivity of cyanophage PP in shallow fresh- water bodies in an extraordinarily fast and effective manner, whereas rapid photoreactivation may contribute to balancing the rapid phototoxicity. KEY WORDS: Cyanophage PP · UV-B · Decay ratio · Photoreactivation repair ratio Resale or republication not permitted without written consent of the publisher INTRODUCTION tion by heat-labile and high-molecular-weight dissolved material, and decay by the ultraviolet component of Cyanophages play an important role in aquatic solar radiation (Wommack & Colwell 2000). Currently, ecosystems, such as maintaining the concentration of solar UV radiation, especially UV-B, is considered to the host, promoting cyanobacteria diversity through be the dominant factor in controlling viral infectivity in gene transfer, and decreasing the transfer of organic seawater and could contribute a decay rate of 0.75 d–1 matter and nutrients to higher trophic levels (Fuhrman to the overall cyanophage decay (Noble & Fuhrman 1999, Weinbauer 2004, Tucker & Pollard 2005). 1997, Garza & Suttle 1998). However, it is known that To understand the role of cyanophages in the ecosys- there is a high abundance and activity of cyanophages tem, we first must understand their behavior in water at the water surface. Therefore, the maintenance of bodies. It is known that the viruses enter the ecosystem virus infectivity despite solar UV damage may rely on by lysing the host, and leave the system by proto- some repair mechanism. There is evidence indicating zoan grazing, attachment to labile colloids, degrada- that photoreactivation repair plays an important role in *Corresponding author. Email: [email protected] © Inter-Research 2007 · www.int-res.com 14 Aquat Microb Ecol 48: 13–18, 2007 restoring the infectivity of viruses and may compen- the bank of Donghu Lake, Wuhan, PR China. Donghu sate for up to 52% of the sunlight-damaged virus Lake is located inside the city of Wuhan and has (Weinbauer et al. 1997). Photoreactivation has been suffered from eutrophication since 1980. The viral studied extensively in bacteriophages and natural samples were filtered through a 0.45 µM filter before virus communities in seawater. However, there have treatment periods as follows: spring, from 08:30 to been no reports regarding photoreactivation in UV-B- 17:30 h on March 6, 2004; summer, from 08:30 to damaged cyanophages, especially cyanophages in 17:30 h on July 7, 2004; autumn, from 08:30 to 17:30 h freshwater. In addition, researchers have previously on October 7, 2004; and winter, from 08:30 to 16:00 h documented that viral abundance can change over a on January 3, 2005. All sampling days were sunny. very short time (Bratbak et al. 1990), implying a rapid During the day of experiments, solar UV-B intensity rate of virus production and removal in aquatic ecosys- was measured hourly with a VLX-3W radiometer tems (Noble & Fuhrman 2000). Thus, it is important to (Vilber Lourmat) at 320 nm. The Secchi disk depth of understand how UV damage and photoreactivation the sampling site was measured via a 25 cm Secchi contribute to the rapid changes in cyanophage levels. transparency dish, and the surface water temperature In our previous work, we found a high abundance was measured directly by a mercury thermometer at and activity of one type of cyanophage in several midday. eutrophic lakes in Wuhan, in the Hubei Province The tested group was treated in the following way: of China. This cyanophage can infect Plectonema triplicate 2.5 ml cyanophage PP suspensions (106 PFU boryanum and Phormidium foveolarum and was ml–1) were placed in small polyethylene (PE) bags in named cyanophage PP (after the respective first letters which UV-B and UV-A were attenuated by 9 and 5%, of its 2 hosts). This virus was characterized as a short- respectively, and the bags were then lowered verti- tailed, icosahedral-shaped, double-stranded DNA cally to water depths of 20, 70, and 120 cm, respec- virus, 52 nm in size and with a genome of 30 kbp (Zhao tively (Garza & Suttle 1998). In the control group, et al. 2002, Guo et al. 2003). In this study, we investi- cyanophage PP was placed in an anti-UV windscreen gated solar UV damage in cyanophages and photore- membrane bag (Model 3535, provided by 3M com- activation repair in the damaged virus in a shallow pany), in which UV-B was attenuated by >99% and freshwater lake, across the 4 seasons of a year. Our incubated in the same way as the tested group. In results indicate that rapid decay of cyanophages spring, summer, and autumn, samples were exposed to induced by solar UV radiation can be compensated for sunlight from 08:30 to 17:30 h, and collected after 3, 6, by rapid photoreactivation by host cyanobacteria. and 9 h exposure. In winter, samples were exposed to These results may help to understand the fluctuations sunlight from 08:30 to 16:00 h, and collected after 3, 6, in levels of cyanophages in freshwater. and 7.5 h exposure. Measurement and calculation of the decay ratio of cyanophage PP and the host’s photoreactivation ratio MATERIALS AND METHODS (repair ratio). The amount of decay was inferred from the difference between the titer of cyanophage PP with Cyanobacterium and cyanophage. The cyanobacte- or without exposure to solar UV-B. The decay ratio (the rial strain, the axenic Plectonema boryanum IU594, percentage of infectivity lost) was denoted as the ratio originally obtained from the Freshwater Algae Collec- (%) between the amount of decay and the titer of the tion of the Institute of Hydrobiology, Chinese Academy initial cyanophage PP in the experiment. The photo- of Sciences, was cultured in AA medium (Richard reactivating level was inferred from the difference be- 1988) at 28°C; 1500 Lux white light was provided by a tween the titer of cyanophage PP, which was exposed white fluorescent tube (Osram L 18W/160) and the to solar UV-B with or without photoreactivation treat- photocycle was set to 16 h light:8 h dark. ment. The repair ratio (the percentage of revived in- Cyanophage PP was prepared as follows: 2 l of Plec- fectivity by photoreactivation) was denoted as the tonema boryanum IU594 culture (107 cells ml–1) was ratio (%) between the amount of photoreactivation and concentrated to 200 ml by centrifugation at 5000 × g amount of decay. for 5 min, and then inoculated with 10 ml of 108 PFU The titer of cyanophage PP was measured under ml–1 cyanophage PP (PFU, plaque-forming unit). After red light (>610 nm), which prevents photoreactivation incubation at 28°C for 24 h, the cyanobacteria were (wavelengths from 300 to 500 nm) (Sinha & Hader completely lysed. Chloroform (10%) was then used to 2002) and which was necessary for the infection of precipitate the host cell debris, and the supernatant hosts by cyanophage PP and for forming the PFU in the was stored at 4°C (Fox et al. 1976). plaque assay (Zhao et al. 2002). Red PE cellophane was Sample treatment in the field. The sample treatment used to screen the light from the white fluorescent site (30° 32.771’N, 114° 21.963’ E) was set to 80 m from tube (Osram L 18W/160), providing red light. Under Cheng et al.: Decay of cyanophage 15 red light conditions, each of the 0.1 ml tested group ) 200 Spring and control group samples were quickly mixed with –2 180 0.9 ml host cells (>107 cells ml–1). The mixture was Summer adsorbed for 1 h, then serially diluted and used for the 160 Autumn plaque assay in the plate (Suttle 1993).