# A Novel Benthic Phage Infecting _Shewanella_ with Strong Replication Ability
## 3 Results and Discussion
### High Replication Capability Reflected by the High Burst Size  
The one-step growth curve of the phage S0112 revealed that its eclipse and latent period of phage infection occurred during 2 h 45 min and 3 h post-infection, respectively (Figure 3), which is much longer than most phages isolated before (Figure 4). We randomly counted sixty-eight phages isolated from aquatic environments infecting strains such as _Roseobacter_ spp., _Vibrio_ spp., _Pseudomonas_ spp., _Aeromonas_ spp., _Acinetobacter_ spp., _Escherichia_ spp., etc., and found that 91% of the viruses have the latent period of less than 2 h (Table S1). Comparatively, the latent periods of the phages which were isolated from seawater or sewage and have similar hosts with that of phage S0112 (i.e., _Shewanella_ spp. strains) are only 12.7-60 min [12; 13; 14]. We speculate that the physiological state or doubling time of bacterial hosts may vary in different environments, which possibly have influences on the phage latent period. The hosts listed in Table S1., such as _Vibrio_ spp., _Escherichia_ spp., and _Acinetobacter_ spp., usually have a rapid doubling time of <30 min, whereas the doubling time of the phage S0112's host _Shewanella indica_ KJW27 is about 70 min. At present, it is not clear about the potential relationship between the host doubling time and the phage latent period, which is worth exploring in the future. Figure 4: The latent period of sixty-eight phages infecting _Roseobacter_ spp., _Vibrio_ spp., _Pseudoalteromonas_ spp., _Aeromonas_ spp., _Acinetobacter_ spp., _Escherichia_ spp., etc. Figure 3: One-step growth curve of _Shewanella_ phage S0112. Open circles represent non-chloroform-treated samples; closed circles represent chloroform-treated samples. Error bars show standard deviations among triplicate samples. Concurrent with the long phage latency, the burst size of phage S0112 was as high as 1170 phage particles per infected cell, which is 5-10 times higher than that of most phages. It has been suggested that the increase of latent period might result in the formation of more progeny phages, i.e., higher burst size [40]. Based on this, it is not abnormal for phage S0112 to have such a high burst size, because the longer latent period can provide ample time for the replication and packaging of progeny phages. Moreover, the host metabolic activity and its survival environment were considered to be closely related to phage latency and burst size [41]. Fast-growing cells under favorable environments can produce more progeny phages than in tough environments [41]. In this study, phage S0112 and its host were all isolated from nutrient-replete coastal sediments. High content of organic matter in coastal surface sediments can boost bacterial growth. When vigorous bacteria are infected by phages, their stronger metabolic activities might provide more energy for viral replication. This is likely why phage release or phage burst size is often higher in laboratory culture than in natural environment. Based on the very limited reports of laboratory-cultured benthic phages, the other two coastal sediment phages, i.e., VpKK5 and AB1, infecting _Vibrio parahaemolyticus_ and _Actinetobacter baumannii_ respectively, also showed relatively higher burst sizes, which is 180 and 409 phage particles per infected cell [18; 19]. In addition, viral production in marine sediments was reported to be higher than in the water column [42]. Although the nutrient-rich coastal sediments are beneficial for bacterial growth, the complexity of sediment components, such as the presence of abundant extracellular enzymes, may be detrimental to phages and promote phage decay and degradation [42]. Therefore, we propose that the strong replication ability of benthic phages might be a survival strategy adapting to the coastal sediment environment to ensure that enough progeny phages can survive the tough conditions.