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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 University, Keelung 202-24, Taiwan; [email protected] 2 Center of Excellence for the , 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 . 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 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 −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 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 . 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 cycling [1], a vital process in sustaining microbial food webs, particularly in Publisher’s Note: MDPI stays neutral oligotrophic . 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 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 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 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 and 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 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 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. * The significance of the difference between the value of in situ and warming incubation. ± represents the SD estimated from triplicate measurements. nd means not detected.

Prokaryotic Viral Prokaryotic Temperature VPR b Burst Size Growth Rate a Production c Mortality c (106 Viruses (105 Cells (Viruses Nov (◦C) (h−1) mL−1 h−1) mL−1 h−1) −1) In situ 22 ± 0.5 0.08 ± 0.06 9.1 ± 1.2 nd nd nd Increased 24 ± 0.5 0.34 ± 0.09 * 10.3 ± 1.6 1.44 0.68 21 temperature Dec In situ 20 ± 0.5 0.06 ± 0.04 13.2 ± 2.6 nd nd nd Increased 23 ± 1.0 0.41 ± 0.05 * 10.6 ± 1.8 0.77 0.56 14 temperature a: Prokaryotic growth was measured during the sampling period in exponential growth phase. b: VPR (virus-to-prokaryotes ratio) in diluted waters was calculated at the end of the experiments. c: Viral production and prokaryotic mortality were estimated using the linear regression between viral and prokaryotic abundance and time between 9 h and 12 h.

2.3. Determination of Prokaryotic Mortality by Viruses and Burst Size Prokaryotic mortality by viruses was measured following the same viral production analysis, where the prokaryotic abundance was monitored over time. Particularly, this was only estimated when prokaryotic abundance significantly decreased during the incubation period (i.e., generally 8 h after the onset of the experiment). Then, the linear regression between prokaryotic abundance and time observed during the time-course experiments was used to calculate prokaryotic mortality by viruses; it was expressed as the number of cells mL−1 h−1. Subsamples were collected at each hour during the incubation time. Furthermore, the burst size was estimated using the results from the time courses; the increase in viral abundance with time was divided by the number of killed prokaryotic cells [14].

2.4. Enumeration of Viral and Prokaryotic Abundance by Flow Cytometry (FCM) Viral subsamples of 0.5 mL were collected every 1 h from each incubation set up and fixed in glutaraldehyde (0.5% final concentration) at 4 ◦C for at least 15 min. Subsequently, the collected subsamples were deep-frozen in liquid . On the other hand, 1-mL of prokaryotic subsamples were also collected every 1 h from each incubation and fixed in Diversity 2021, 13, 409 4 of 8

paraformaldehyde (1% final concentration). The collected viral and prokaryotic samples were preserved at a temperature of −80 ◦C until the flow cytometry (FCM) analysis. Viral and prokaryotic samples were analyzed using a CytoFLEX S flow cytometer (Beckman Coulter, Indianapolis, IN, USA) with a 488 nm air-cooled argon- laser, a standard 525 nm filter, and an SYBR signal trigger. Prior to , viral samples were diluted at 1:10 in TE buffer (pH 8.0, EM grade) to minimize the interference from high abundance. The diluted samples were stained with SYBR Green I (final concen- tration 1:50,000 of commercial stock) and incubated for 10 min at 80 ◦C without light. After staining, these were then cooled to 25 ◦C in an ice bath and processed through FCM according to Brussaard’s method [15]. TE buffer’s blank controls stained with the same concentration of SYBR Green I were run to detect and eliminate any noise from the buffer. The quantification of viruses by FCM is comparable to epifluorescence microscopy and is suitable for fast quantification [16]. Furthermore, the prokaryotic samples were pigmented with an SYBR Green I (final concentration 1:10,000) in the dark for 15 min and processed through FCM according to the protocol of Hammes and Egli [17].

2.5. Statistical Analysis The relationship between viral abundance and the time for triplicate incubations was analyzed using linear regression analysis. Meanwhile, an analysis of variance (ANOVA) was used to test the significance of the slope. Warmer conditions and ambient were compared using the nonparametric Mann-Whitney test, while The STATISTICA 7.0 software was used for all statistical operations. Notably, a probability value of <0.05 was considered significant.

3. Results In the study, in situ prokaryotic and viral abundance were 4.0 ± 0.6 × 105 cells mL−1 and 5.9 ± 0.9 × 106 viruses mL−1 in November, and 1.6 ± 0.4 × 105 cells mL−1 and 4.3 ± 0.3 × 106 viruses mL−1 in December, respectively. The time-course experiments in this study revealed an increased prokaryotic abundance in the first 6 or 7 h, maintained the higher values for 3–4 h, which subsequently decreased (Figure1A,B). Furthermore, during the sampling period in the exponential growth phase, the prokaryotic growth rate was 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 during the winter seasons, respectively (Table1). Significantly increased prokaryotic growth rates resulted from the warming conditions (Mann-Whitney test, p < 0.05) (Table1). In this study, temporal patterns exhibited a change in viral abundance with incu- bation time between in situ and warmer conditions in the cold seasons (t-test, p < 0.05) (Figure1C,D ). In the in-situ temperature incubation, the viral abundance fluctuated be- tween 1.9 and 2.7 × 106 viruses mL−1 in November and 0.9 and 1.6 × 106 viruses mL−1 in December (Figure1C,D). In contrast to in situ temperature incubation, viral abundance increased abruptly at 12 h and 10 h during the warming incubation in November and December, respectively (Figure1C,D). In the current study, a significantly increased viral abundance with incubation time after 9 h was observed in warmer conditions (Figure1C,D). Thus, the VP rates were estimated from the viral abundance’s slope of the incubation experiment between 9 h and 12 h (Figure2A) to compare the difference between in situ and warmer conditions in the colder months. Obviously, in the colder months, the overall value of VP was about 0.77–1.44 × 106 viruses mL−1 h−1 under warming experiments (Table1). However, no significant slope of the regression line was observed in ambient temperature for 12 h incubation, and we did not estimate the VP under ambient treatments in this study (ANOVA, p > 0.05) (Table1). Diversity 2021, 13, x FOR PEER REVIEW 5 of 8

1.44 × 106 viruses mL−1 h−1 under warming experiments (Table 1). However, no significant slope of the regression line was observed in ambient temperature for 12 h incubation, and we did not estimate the VP under ambient treatments in this study (ANOVA, p > 0.05) (Table 1). The prokaryotic mortality by viruses and burst size are reported in Table 1. In this study, we estimated prokaryotic mortality using the linear regression between prokary‐ otic abundance and time between 9 h and 12 h (Figure 2B). The prokaryotic effects of viral infection increased along with an increase in temperature, accounting for 0.68 and 0.56 × 5 −1 −1 Diversity 2021, 13, 409 10 cells mL h in November and December in warmer conditions, respectively (Table5 of 8 1). Furthermore, based on our estimation, the burst size of 21 and 14 gives a rough esti‐ mate in this study (Table 1).

Diversity 2021, 13, x FOR PEER REVIEW 6 of 8 FigureFigure 1. Temporal 1. Temporal variations variations in prokaryotic in prokaryotic and viral and abundance viral abundance at ambient at ambient (), and ( warming■), and temperaturewarming temperature treatments ( ) in Novembertreatments ( A(○,C)) in and November December ( (AB,CD)), and respectively. December Vertical (B,D), bars respectively. show ± standard Vertical deviations bars show (n =± 3).standard devia‐ # tions (n = 3).

Figure 2. 2. RelationshipsRelationships between between viral viral abundance abundance (A), prokaryotic (A), prokaryotic abundance abundance (B), and (incubationB), and incubation △ timetime between between 9 9h hand and 12 12 h hin inNovember November ( ()4 and) and December December (+). (—) (+). and (—) (– and –) are (– –) regression are regression lines lines for for November and December. November and December. 4. Discussion Some models on global climate change predicted a +2 °C increase in temperature increase over the next century [10]. Thus, our experimental scales on warming treatment were reasonable with this study’s temperature change rates. The favorable impact of the small and incremental increases in temperature on prokaryotic growth rates observed in this study is consistent with those in prior studies [4,18]. For one, Li [19] suggested that temperature dominantly affects prokaryotic growth rates in colder waters for a year. Meanwhile, our findings demonstrate that possible temperature changes in the Pacific coastal waters may result in increased prokaryotic activity in the future, which will promote other microbial activities. However, previous studies reported that due to counteracting effects (e.g., elevated predation), an increase in abundance might not nec‐ essarily follow after a warming stimulation of prokaryotic growth [20]. Further, a few studies have investigated the temperature’s effects on carbon transfer between prokary‐ otes and in natural microbial communities. Notably, they reported positive ef‐ fects of temperature on the nanoflagellates’ grazing rates on prokaryotes [4]. In the pre‐ sent study, the size fractionation used for the grazers (<2 μm) was employed to eliminate nanoflagellates. While we did not directly assess prokaryotic grazing rates in our ex‐ periments, nanoflagellate grazing rates seemed higher in the warming experiments during winter than ambient temperature treatment in our study environment [21]. The present study mainly aimed to observe the effect of the increase in temperature on the prokaryotic loss caused by viruses and the viral ecology in the Pacific coastal wa‐ ters. The results of the warming experiments confirmed the hypothesis that prokaryotic and viral abundance increase with rising temperatures. The virus dilution technique is preferred to determine marine viral production [10]. Particularly, a dilution of viral

Diversity 2021, 13, 409 6 of 8

The prokaryotic mortality by viruses and burst size are reported in Table1. In this study, we estimated prokaryotic mortality using the linear regression between prokary- otic abundance and time between 9 h and 12 h (Figure2B). The prokaryotic effects of viral infection increased along with an increase in temperature, accounting for 0.68 and 0.56 × 105 cells mL−1 h−1 in November and December in warmer conditions, respectively (Table1 ). Furthermore, based on our estimation, the burst size of 21 and 14 gives a rough estimate in this study (Table1).

4. Discussion Some models on global climate change predicted a +2 ◦C increase in temperature increase over the next century [10]. Thus, our experimental scales on warming treatment were reasonable with this study’s temperature change rates. The favorable impact of the small and incremental increases in temperature on prokaryotic growth rates observed in this study is consistent with those in prior studies [4,18]. For one, Li [19] suggested that temperature dominantly affects prokaryotic growth rates in colder waters for a year. Meanwhile, our findings demonstrate that possible temperature changes in the Pacific coastal waters may result in increased prokaryotic activity in the future, which will promote other microbial activities. However, previous studies reported that due to counteracting effects (e.g., elevated predation), an increase in abundance might not necessarily follow after a warming stimulation of prokaryotic growth [20]. Further, a few studies have investigated the temperature’s effects on carbon transfer between prokaryotes and protists in natural microbial communities. Notably, they reported positive effects of temperature on the nanoflagellates’ grazing rates on prokaryotes [4]. In the present study, the size fractionation used for the grazers (<2 µm) was employed to eliminate nanoflagellates. While we did not directly assess prokaryotic grazing rates in our experiments, nanoflagellate grazing rates seemed higher in the warming experiments during winter than ambient temperature treatment in our study environment [21]. The present study mainly aimed to observe the effect of the increase in temperature on the prokaryotic loss caused by viruses and the viral ecology in the Pacific coastal waters. The results of the warming experiments confirmed the hypothesis that prokaryotic and viral abundance increase with rising temperatures. The virus dilution technique is preferred to determine marine viral production [10]. Particularly, a dilution of viral abundance “ensures the low occurrence of new phage infections and reduces possible viral losses due to new infection” [22]. Our results observed that the rise in sea surface temperature caused by global warming could significantly affect the viral population and their interaction with the marine microbial community. At the same time, studies reported that warming stimulates prokaryotic growth rates and viral production [23–26]. Noteworthily, although these studies focused mainly on single-specie host cultures and viral isolates, there is little knowledge on the effects of warming on the viral dynamics of more complex marine environments. One limitation of this study is the inability of prokaryotic communities in the short experimental time to properly simulate the effect of long-term adaptation likely occurring in natural systems. Thus, the results may be biased. However, in order to interpret the microbial ’s response to environmental stressors, the warming experiment was used. The data of the experiment show that warming (+2 ◦C) enhanced VP in this study. Indeed, we think that variations of prokaryotic growth and VP are subjected to increasing temperature. Previous studies reported that while the temperature can promote the enhancement of prokaryotic production and growth rates [27], it can “also shorten virus latent period, and increase burst size”, which increases viral production [23,24]. The shortened virus latent period may induce higher VP in the warming experiments, which could explain the overall increased VP in the warmer experimental treatments compared to the in situ condition. Interestingly, there was no significant difference in VPR (virus- to-prokaryotes ratio) in our study between in situ and warmer conditions at the end of incubation experiments (Table1). Recent findings demonstrated how phages infect their Diversity 2021, 13, 409 7 of 8

prokaryotic hosts differently, “causing lysis at high temperatures and lysogeny at lower temperatures” [28]. As a result, variations in daily temperature would lead to “switches in phage-prokaryotes interactions” [28].

5. Conclusions In conclusion, based on these short-term experimental results, and allow us to hy- pothesize that climate-change-induced warming can be expected to affect prokaryotic and viral activities significantly. Our results provide evidence that the effects of rising temperatures resulted in increased viral production and found that it can exert significant effects on prokaryotic mortality under the global warming scenario. Moreover, this study also implied that warming water temperature reduces the transfer efficiency from prokaryotes to higher trophic levels in coastal waters during cold seasons.

Author Contributions: Conceptualization: A.-Y.T.; methodology: A.-Y.T.; validation: A.-Y.T.; formal analysis: A.-Y.T. and G.-C.G.; investigation: A.-Y.T.; resources: A.-Y.T. and G.-C.G.; data curation: A.-Y.T.; writing–original draft preparation: A.-Y.T. and V.M.; writing–review and editing: A.-Y.T. and V.M.; funding acquisition: A.-Y.T. and V.M. All authors have read and agreed to the published version of the manuscript. Funding: The research was conducted in the frame of the Russian state assignment No. 121040600178- 6 and supported by RFBR projects 18-44-920026 (works on the GAF phenomenon) and 21-55-52001, and the Ministry of Science and Technology, ROC (Taiwan), grant number NSC 109-2611-M-019-013. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: We appreciate the language editing and helpful comments related to this manuscript from Choice Language Service. Conflicts of Interest: The authors declare no conflict of interest.

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