diversity Brief Report Experimental Warming Effects on Prokaryotic Growth and Viral Production in Coastal Waters of the Northwest Pacific during the Cold Season An-Yi Tsai 1,2,*, Gwo-Ching Gong 1,2 and Vladimir Mukhanov 3 1 Institute of Marine Environment and Ecology, National Taiwan Ocean University, Keelung 202-24, Taiwan; [email protected] 2 Center of Excellence for the Oceans, National Taiwan Ocean University, Keelung 202-24, Taiwan 3 A.O. Kovalevsky Institute of Biology of the Southern Seas, Russian Academy of Sciences, 299011 Sevastopol, Russia; [email protected] * Correspondence: [email protected]; Tel.: +886-2-2462-2192 (ext. 5705); Fax: +886-2-2462-0892 Abstract: Climate warming can directly affect biological processes in marine environments. Here, we investigated if warming (+2 ◦C) can change dynamics in viral and prokaryotic populations in the cold seasons in natural seawaters. We monitored the changes in viral production and prokaryotic growth rate. The prokaryotic average gross growth rates were 0.08 and 0.34 h−1 in November and 0.06 and 0.41 h−1 in December in the in situ and warming experiments, respectively. We found that warming water temperature resulted in a significant increase in prokaryotic growth rates. In warming experiments, the overall viral production rate was about 0.77–14.4 × 105 viruses mL−1 h−1, and a rough estimate of prokaryotic mortality was about 5.6–6.8 × 104 cells mL−1 h−1. Based on our estimation, burst sizes of about 21 and 14 viruses prokaryotes−1 were measured under the Citation: Tsai, A.-Y.; Gong, G.-C.; experimental warming period. Moreover, the results found that an increased water temperature Mukhanov, V. Experimental Warming in the subtropical western Pacific coastal waters increases prokaryotic growth rates, enhances viral Effects on Prokaryotic Growth and production, and changes the carbon fluxes in the trophic interactions of microbes. Viral Production in Coastal Waters of the Northwest Pacific during the Keywords: warming; viral production; prokaryotic growth rate; prokaryotic mortality; viral lysis Cold Season. Diversity 2021, 13, 409. https://doi.org/10.3390/d13090409 Academic Editors: Hera Karayanni, 1. Introduction Cinzia Corinaldesi and Michael Wink Viruses are an integral part of the microbial community, substantially causing mortality Received: 31 July 2021 among marine prokaryotes. According to the principle of the “Killing The Winner” model, Accepted: 26 August 2021 changes in the prokaryotic community’s structure manifest the ecological impacts of viral Published: 27 August 2021 lysis [1]. They also play a crucial function in the operations of marine food webs and nutrients cycling [1], a vital process in sustaining microbial food webs, particularly in Publisher’s Note: MDPI stays neutral oligotrophic ecosystems. with regard to jurisdictional claims in Temperature is an important environmental factor that influences prokaryotic growth published maps and institutional affil- efficiency [2], and growth rate [3,4]. Changes in prokaryotic growth were expected to affect iations. microbial processes (e.g., viral lysis and prokaryotic mortality rates). Several field studies have further evidenced an increase in the rates of viral lysis among phytoplanktons over the North Atlantic Ocean from high to low latitudes, which, interestingly, is positively correlated with temperature [5]. Changes in host physiology caused by temperature Copyright: © 2021 by the authors. were indeed found altering the mechanisms of viral lysis, which possibly engenders Licensee MDPI, Basel, Switzerland. the development of viral resistance [6]. The rise of sea surface temperature caused by This article is an open access article global warming may significantly impact the viral population and their interaction with distributed under the terms and the marine microbial community [7]. The mechanisms of how global warming affects conditions of the Creative Commons microbial communities and viruses remain in question. There is still limited knowledge on Attribution (CC BY) license (https:// the effect of warming on prokaryotic mortality by viruses and even less information about creativecommons.org/licenses/by/ the prokaryotes-viruses interaction. 4.0/). Diversity 2021, 13, 409. https://doi.org/10.3390/d13090409 https://www.mdpi.com/journal/diversity Diversity 2021, 13, 409 2 of 8 We have performed several studies about prokaryotic growth on the subtropical western Pacific coastal waters [8,9]. These waters have been described in our previous reports derived from data collected from 1999 to 2001 [8]. A previous study found surface water temperatures in March reaching about 15–16 ◦C and gradually rising to 29 ◦C by the month of July in the subtropical western Pacific coastal waters. The monthly average concentration of nitrate is the lowest between June and October (>1 µM), when it may reach 12 µM from November to May [8]. Few studies have reported the prokaryotic effects of nanoflagellates and viruses in such a marine environment [9]. Recently, we found a significantly higher occurrence of viral lysis than prokaryotic mortality because of nanoflagellates grazing during cold seasons [9]. Viruses have a crucial role in the aquatic microbial food web by recycling large amounts of carbon and nutrients in winter and preventing prokaryotic production in higher trophic levels [9]. Tsai et al. [8] also reported a seasonal cycle with two phases, namely warm season (>25 ◦C) and cold season (<25 ◦C), having a 10-fold variation of prokaryotic growth. The prokaryotic growth rate and temperatures exceeding 25 ◦C had no significant correlation. Hence, the possibility of changes at the food web’s base (due to increased water temperature) significantly affecting different aspects of marine ecosystems’ operations and structure. To better understand the global nutrient and carbon cycle in seawaters, it is particularly important to examine the impact of increased temperatures on microbial communities. After a century of global warming, large portions of ocean surface waters would incrementally increase to an average temperature of 2 ◦C in approximation [10], most likely leading to structural and functional changes of marine ecosystems. In this study, we examined the impact of a temperature increase of 2 ◦C on viral production during colder seasons. We hypothesized that a temperature-driven increase in growth rates of prokaryotes would enhance viral production, indicating that temperature rise has a different effect on the viral lysis of prokaryotes in the colder months (November and December 2020). In this study, Wilhelm et al.’s virus dilution technique [11] was used to determine the rates of viral production, through which we could estimate the prokaryotic mortality by viruses and burst size in this study. 2. Materials and Methods 2.1. Sampling Samples were collected in November and December 2020 from the surface waters at an established station located in Taiwan’s northeastern coastal waters (25◦09.40 N, 121◦46.30 E). For each sampling, a bucket was used to collect seawater from 07:00 to 08:00 h in the morning (local time). Then, the seawater was gently poured into a clean 5-L Niskin bottle for dilution experiments, and the water’s temperature was measured at the time of casting. Finally, all samples were delivered to the lab immediately within 30 min from the time of sampling. 2.2. Viral Production and Prokaryotic Growth Rate Experiments First, the grazing-free whole water was prepared, gently vacuum-filtering 2 L of surface seawater through a 47-mm diameter and a 2 µm pore-size polycarbonate track- etched filter membrane (Whatman). 500 mL of virus-free water was produced for viral dilution by filtering grazer-free seawater through a Minimate TFF Capsule (Pall), with a 30-kDa molecular weight cut-off. Removal of viruses through 30 kDa TFF does not fundamentally change the carbon and nutrient composition and prokaryotic assemblage in water. Thus, with slight manipulations, this method is suitable for virus dilution studies [12]. Dilution was performed by adding 400 mL of the virus-free water to 100 mL of grazer-free water, decreasing the prokaryotic and viral abundance to approximately 20% to that of the original seawater [11]. The diluted incubation water was thoroughly mixed and filled in 50 mL plastic incubation tubes. All treatments were incubated in a water bath set at the original temperature where the seawater was during sampling. Further, the experimental warming temperature was set at 2 ◦C above in situ values (Table1) . Diversity 2021, 13, 409 3 of 8 Then, the bottles were immediately moved outside the laboratory after preparation to a location near the sampling site. They were then incubated for 12 h under natural light in a thermo-controlled incubator. Particularly, the treatments were performed in triplicates. To determine prokaryotic and viral abundance, 1-mL subsamples were taken every 1 h for a 12 h period at the onset of the experiment. Further, the linear regression between viral abundance and incubation time was used to calculate viral production (VP) (viruses mL−1 h−1). Notably, there was a significant linear relationship between viral abundance and incubation time, while VP was defined as the regression slope [13]. In addition, the growth rate of prokaryotes was estimated in these 20% diluted samples in the exponential growth phase, where the prokaryotic abundance was monitored over time, and the prokaryotic growth rate changes were compared with the in situ and experimental warming temperature samples. The prokaryotic growth rate was calculated as follows: µ = ln (Nt/N0) t −1 where µ is growth rate (h ), N0 and Nt are the prokaryotic abundance at the beginning and the peak of the exponential growth phase, and t is the incubation time that arrival the peak of the exponential growth phase (h). Table 1. Water temperature for the incubation experiments. Prokaryotic growth rate, viral production, number of lysed prokaryotes, and burst size in in situ and warming incubation. The VPR (virus-to-prokaryotes ratio) in diluted waters was calculated after the experiments.