Production of mycosporine-like amino acids of in situ phytoplankton community in Kongsfjorden, Svalbard, Arctic

Journal of Photochemistry and Photobiology B: Biology 114 (2012) 1–14

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Production of mycosporine-like amino acids of in situ phytoplankton community in Kongsfjorden, Svalbard, Arctic Sun-Yong Ha a, Young-Nam Kim c, Mi-Ok Park b, Sung-Ho Kang c, Hyun-choel Kim c, Kyung-Hoon Shin a,⇑ a

Hanyang University, Department of Marine Environmental Science, 1271-3 dong, Sangnok-gu, Ansan, Kyeonggi-do 425-791, South Korea Pukyong National University, Department of Oceanography, 559-1, Daeyeon 3 dong, Nam-Gu, Busan 608-737, South Korea c Korea Polar Research Institute (KOPRI), Division of Polar Climate Research, Get-Pearl Tower, 12 Gaetbeol-ro, Yeonsu-Gu, Incheon 406-840, South Korea b

a r t i c l e

i n f o

Article history: Received 27 October 2011 Received in revised form 23 February 2012 Accepted 27 March 2012 Available online 5 April 2012 Keywords: UV-absorbing compounds Mycosporine-like amino acids Thalassiosira sp. Phaeocystis sp. Carbon stable isotope Kongsfjorden

a b s t r a c t The spatial distribution of UV-absorbing compounds (mycosporine-like amino acids, MAAs), was investigated by comparing the phytoplankton community structures in the inner and outer waters of the Kongsfjorden inlet, which is located in arctic Svalbard. Thalassiosira sp. and Phaeocystis sp. were dominant in the outer waters of the Kongfjorden inlet, demonstrating high chlorophyll a (chl a) concentrations and low MAA concentrations in the outer bay waters. However, Kongsfjorden Bay was dominated by Phaeocystis sp. and demonstrated high MAA concentrations despite low chl a concentrations. The carbon fixation rate at a station located inside Kongsfjorden Bay (T05) was significantly photo-inhibited by UV radiation, demonstrating higher production rates of MAA and chl a than at a station (B09) in outer bloom waters. Additionally, the turnover rates of individual MAAs were faster inside the Kongsfjorden Bay than in the outside waters. As a result, the natural phytoplankton community demonstrated different UV adaptation mechanisms according to the phytoplankton species, in this case, Thalassiosira sp. vs. Phaeocystis sp. It is possible to understand real-time changes for newly photosynthesized MAAs as UV-absorbing compounds in the natural phytoplankton community. This takes place via determination of in situ MAA production rates using 13C tracer and High Performance Liquid Chromatography (HPLC) combined with an isotope ratio mass spectrometer (irMS). Ó 2012 Elsevier B.V. All rights reserved.

1. Introduction With depletion of the ozone layer in the Arctic atmosphere [1,2], the transmission of ultraviolet (UV) radiation has also increased, thereby affecting aquatic organisms in high latitude marine ecosystems [3,4]. The increased destruction of the ozone layer in the Northern Hemisphere [5,6] has also influenced the growth of phytoplankton or macroalgae in Arctic waters through enhanced exposure to UV radiation [3,7–9]. According to research conducted in Kvalsund, Norway, UV radiation inhibits the efficiency of phytoplankton photosynthesis by approximately 50% [10], and the primary productivity of phytoplankton in Kongsfjorden is reported to be decreased by about 0.05–4.1% due to UV radiation [6]. However, studies on the effects of UV radiation on primary productivity of phytoplankton through in situ incubation have primarily been conducted in the southern oceans [11–13], while studies conducted in the Arctic are scarce [6,7]. According to recent research, UV radiation inhibits photosynthesis and the primary productivity of phytoplankton [14,15] and

⇑ Corresponding author. Tel.: +82 (0)10 8243 6637; fax: +82 (0)31 416 6173. E-mail address: [email protected] (K.-H. Shin). 1011-1344/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jphotobiol.2012.03.011

causes adverse effects on cellular protein synthesis and DNA [16,17]. Moreover, UV radiation interferes with the absorption of nutrients [18] and affects the biochemical processes of various types of cellular enzymes [19,20]. The effects of UV radiation on the metabolism or survival of cells vary significantly among different species of phytoplankton because the ability of phytoplankton to adapt to light upon exposure to UV radiation and to repair damaged DNA are variable depending on the species [16,21]. Meanwhile, organisms have survived in extreme environments by manipulating their responses to strong light or UV radiation via production of a variety of photoprotective compounds [22]. Similarly, phytoplankton protects themselves from UV radiation through secretion of UV-absorbing compounds that can block harmful UV radiation or by releasing photoprotective pigments [15]. In particular, UV-absorbing compounds known as mycosporine-like amino acids (MAAs) have the ability to protect organelles from harmful UV radiation and are generally found in aquatic organisms including marine primary producers [23,24], freshwater primary producers [25–28], and cyanobacteria [29]. MAAs are small, water-soluble compounds with a molecular weight less than 400 Da and are composed of aminocyclohexenon or aminocycloheximine rings containing amino alcohol substituents [23] Additionally, MAAs have an absorption maximum at 310–360 nm

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[23,30,31] and are synthesized in phytoplankton and the cells of other aquatic organisms to provide important photoprotective effects when exposed to harmful wavelengths [32]. Several studies have been conducted to understand the production of MAAs following the exposure of a single species of phytoplankton to artificial UV radiation [15,33,34], to understand the distribution of MAA concentrations within in situ POM or DOM [21], and to understand the MAA induction process using mesocosms [35,36]. Kongsfjorden is an Arctic glacier fjord area on the western coast of Spitsbergen island of Svalbard, which is located at the highlatitude Arctic region (79°N and 12°E), but it exhibits characteristics of a subarctic environment due to the effects of the high temperature Atlantic Gulf Stream. There is no submarine sill disturbing the flow of currents at the entrance of Kongsfjorden. Thus, the inflow of offshore water is unobstructed, while the flow of glacial outflow is also substantial, causing a characteristically active physical change between the Svalbard coastal water (glacial outflow) and western continental shelf coastal water (inflow of offshore water). Moreover, the surface water of Kongsfjorden varies according to complex factors including tides, currents, winds, precipitation, water inflow from glacial melt and the coast, and sea ice/ glacier drift. All of these factors act differently depending on the day or the season; thus, the characteristics of the surface water show daily or seasonal changes [37]. These changing variables in the physical environment are observed alongside changes in the community structure of phytoplankton [38], resulting in characteristics that are distinct from phytoplankton communities in offshore water [39]. In the present study, the spatial distribution patterns of MAAs (UV-absorbing compounds) in phytoplankton communities were determined based on samples from within Kongsfjorden Bay and offshore waters. Additionally, total carbon fixation rates and production rates of individual MAAs were determined with in situ incubation experiments using a 13C tracer. The objective of present study is to understand the production processes of MAAs as a bloom survival strategy for marine phytoplankton community in subarctic regions, where receives intensified exposure to UV radiation in the spring. 2. Materials and method 2.1. Study area Sampling and in situ incubation experiments were conducted in waters around the Arctic Svalbard coast of Kongsfjorden Bay from May 22–29, 2009 (Fig. 1). During the R/V FARM cruise track, seawater samples were collected at 25 sampling stations located throughout offshore waters with relatively high phytoplankton biomass and in waters adjacent to the sea ice of the Kongsfjorden inland sea. Phytoplankton species composition, including identification and quantitative analysis, was determined at five stations (B01, B03, B09, A05, and T05). The distribution of phytoplankton species composition in seawater was determined using microscope by the Korean Polar Research Institute (KOPRI). Surface water samples were filtered in triplicate 1 L at a time using pre-baked (450 °C) GF/F filter paper to measure MAA concentrations and then stored at 80 °C. Samples were transported to the laboratory using liquid nitrogen canisters. 2.2. In situ culture experiments using a

station (T05) dominated by Phaeocytis sp. in Kongsfjorden Bay. The ocean collar images of ocean color data of MODIS (Moderate Resolution Imaging Spectroradiometer, which is board on AQUA satellite) confirmed the progress of phytoplankton bloom occurrence from 22 May to 29 May, 2009 (Fig. 2). The AWIPEV Arctic Research Base at the Alfred Wegener Marine and Polar Institute (AWI) provided ultraviolet irradiation data during the in situ incubation period. Average UV intensity was calculated at 13.2 W m2. The study area was divided into three groups: inner bay, outer bay, and bloom areas specified by the Aqua MODIS satellite. From the MODIS the bloom had occurred since May 24th (Fig. 2e) and the bloom became larger on May 29th 2009 (Fig. 2f). Surface waters at sampling stations B09 and T05 were collected and transferred into quartz (HanJin Quartz Co.) and polycarbonate bottles (PC; NalgeneÒLabware) for in situ incubation analysis. Quartz and PC bottles are known to have different light cutoff wavelengths. The PC bottles, which block UV radiation (photosynthetically available radiation, PAR + UVA exposure; PA), were used as a control for comparison with quartz bottles, which transmit UV radiation (PAR + UVA + UVB exposure; PAB) (Fig. 3). NaH13CO3 (99%), which was added as a tracer, increased up to 15% of the 13 C in the total DIC concentration pool. To observe the effects of UV radiation, the incubation bottles were then immersed in in situ seawater on the deck at sampling stations B09 and T05, where the bottles remained incubated for 72 h. The UV-exposing quartz bottles and the UV-blocking PC bottles were split into two sets and incubated at the same time for duplication purposes. After the incubation was complete, 1 L of incubated sample was filtered at a time using pre-baked GF/F filter paper, and a liquid nitrogen canister was used to transport the samples to the laboratory. The filter samples were stored at 80 °C until analysis. 2.3. Carbon fixation rate To determine the carbon fixation rate relative to photosynthesis, NaH13CO3 (99%) was added to the seawater and incubated for 72 h. Then, 500 mL of sample was filtered with GF/F 25-mm filter paper (pre-baked at 450 °C for 4 h) and stored at 80 °C until analysis. The filtered sample was completely dried in a lyophilizer and exposed to 1 N HCl fumes overnight to remove inorganic carbon. Neutralization was then performed using NaOH fumes. The percentage of enriched 13C atoms was measured using an EA-irMS (EuroEA-Isoprime IRMS, GV Instruments, UK), and the carbon fixation rate was calculated using the following equation [40].

Carbon uptake rateðqcðtÞÞ ¼

DPOCðtÞ ais  ans POCðtÞ ¼  t t aic  ans

ð1Þ

where ais is the 13C% of particulate organic carbon in an incubated sample; ans the 13C% of particulate organic carbon in a natural (non-incubated) sample; aic the 13C% of dissolved inorganic carbon in the incubation bottlet: incubation time POC(t) is the concentration of particulate organic carbon in the incubated sample. Photosynthetic inhibition under UV radiation (i.e., carbon fixation in the PAB treatment relative to that in the PA control) during the incubation period was calculated as: UV-B inhibition = ((CPA  CPAB)/(CPA)  100, where CPA and CPAB are the carbon fixation values in the PA and PAB treatments, respectively. 2.4. Extraction and analysis of UV-absorbing compounds (mycosporine-like amino acids)

13

C tracer

To determine total carbon fixation and production rates for MAAs during natural UV radiation exposure, in situ incubation experiments were conducted at an offshore station (B09) where blooms occur and then compared with those at an inner water

To analyze MAAs in particulate organic matter, 1 L of each incubated water sample was filtered through pre-baked GF/F 47-mm filter paper and stored at 80 °C until analysis. Three mL 100% MeOH was added to the filtered sample, and then 30 s in an ultrasonicator (30 s, 50 W; Ulsso Hi-tech ULH-700s) was used to break

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Fig. 1. Locations of sampling stations and in situ incubation sampling stations in Kongsfjorden Bay and offshore, Svalvard.

down the sample. The sample was refrigerated at 4 °C overnight and then extracted. After extraction, a 0.2-lm syringe filter (PTFE 0.20 lm Hydrophobic) was used to transfer the sample to a 2 mL microtube, and the chl a was measured using the Porra [41] method. After measuring the sample, a centrifugal evaporator (EYELA, CVE-200D) was used to completely dry the sample. The dried sample was then dissolved in 500 lL distilled water, 100 lL chloroform was added, and the sample was centrifuged for 10 min at 10,000 rpm. A total of 400 lL supernatant was separated, and the separated material was then injected into a High-Performance Liquid Chromatography (HPLC) to quantitatively analyze the MAAs. MAA analysis was performed using an HPLC system (Agilent Technologies 1200 series; column: Waters 120DS-AP (5 lm) 150 mm  4.6). The detector was an Agilent DAD (G1315D) at 313 nm (250–750 nm scan), and each MAA compound (shinroine

(SH), palythine (PA), porphyra-334 (PR), mycosporine–glycine (MG), and asterina-330 (AS)) was split using a fraction collector (Agilent analyte (G1364C) FC) (Fig. 4). The mobile phase (100% distilled water with 0.1% acetic acid) was used at a constant flow at 0.8 mL/min. Shinorine and porphyra-334 were used as standard reference compounds for quantitative analysis of MAAs. To determine the production rate of each MAA, the MAA compounds were collected through a tin cap (including the pre-baked filter paper). Once the solvents were completely removed, the 13C value of each compound was measured using EA-irMS. Production rate of each MAA was calculated form each 13C atomic percent and concentration using the modified equation proposed by Hama et al. [40,42–45]. The production rates of individual MAAs were calculated using a modified equation [40], and the chl a specific production rate of

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Fig. 2. Distribution of chl a concentrations around Kongsfjorden in the subarctic ocean as captured by the Aqua MODIS Satellite from May 7 to May 29. (a) May 7, (b) May 8, (c) May 9, (d) May 13, (e) May 24, and (f) May 29. Dotted square indicates study area.

S.-Y. Ha et al. / Journal of Photochemistry and Photobiology B: Biology 114 (2012) 1–14

Fig. 2 (continued)

5

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60

incubated sample; ans the 13C atom% in each MAA of natural sample; aic the 13C atom% in 13C enriched inorganic carbon; MAC is the concentration of each MAA carbon at the end of incubation The turnover rate of each MAA is also calculated by the following equation in order to understand the relationship between each MAA concentration and production rate [40]

40

Turnover rateðtÞ ¼

100

Transmission (%)

80

ð3Þ

The possible isotopic discrimination against 13C during photosynthetic uptake was not considered in this study because its correction has little significant effect on the uptake rate [40].

20 0 Quartz bottle PC bottle

-20

ðais  ans Þ 1  ðaic  ans Þ t

300

400

500

600

2.5. Statistical analysis

700

Wavelength (nm) Fig. 3. Light transmission along the wavelength range by polycarbonate and quartz bottles used in the in situ incubations. There are also differences in the transmission of PAR (400–700 nm) for both materials (15% less in PC bottles).

each MAA was obtained by normalization of each chl a concentration. Turnover rates of individual MAAs were calculated using the production rates and concentrations of MAAs.

Mean values and standard deviations of all samples were obtained after calculating the values of duplicates. Statistical significance (p < 0.05) of all samples was obtained through two-way analysis of variance (ANOVA) using Tukey’s test. The correlation coefficient for each individual MAAs compounds and phytoplankton species was obtained through SPSS program version 18 for Windows (SPSS). 3. Result

ð2Þ

3.1. Distributions of chl a and MAA concentrations in Kongsfjorden Bay and in offshore waters

where DMAC is the amount of each MAA carbon photosynthetically produced during the incubation; ais the 13C atom% in each MAA of

The highest concentration of chl a was calculated at 6.8 lg/L (St. B05) in the outer bay waters where the phytoplankton bloom

DMACðtÞ ¼ MAC 

ais  ans aic  ans

Fig. 4. A typical HPLC-DAD chromatograms and absorption spectra of individual MAAs in the present study at station B09; 1: shinorine (kmax: 334); 2: palythine (kmax: 320); 3: porphyra-334 (kmax: 334); 4: mycosporine–glycine (kmax: 310); 5: asterina-330 (kmax: 330).

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occurred, whereas the lowest concentration was 0.15 lg/L in the inner bay waters (St. A03). Average chl a concentration from the bloom area (3.9 (±2.23) lg/L) was higher than those in other areas (inner bay and outer bay). The lowest average chl a concentration was 0.43 (±0.19) lg/L, found in the inner bay. The outer bay area demonstrated a mid-range value (0.93 (±0.58) lg/L), which was indirectly impacted by the phytoplankton bloom. At stations A06 and A07, in particular, higher chl a concentrations were recorded compared with those of other nearby stations (Table 1). The general trend of chl a concentration represented a gradual increase from Kongsfjorden Bay toward offshore waters. The spatial distribution of MAAs demonstrated relatively high concentrations in the inner bay and bloom waters and relatively low concentrations in the middle of Kongsfjorden Bay and offshore waters. The distribution pattern is considered to be a result of changes in the physical environment (seawater temperature) due to the current flowing through the west side of Spitsbergen (Fig. 1 and Table 1) [38]. The highest total MAA concentration value at 25.5 (±1.4) lg/L (Station A1) was recorded in the inner bay, and the lowest MAA concentration value at 3.1 (±0.6) lg/L (Station T03) was recorded at the edge of the sea ice in the inner bay. The highest and lowest MAA concentration values were shown at the inner bay. However, the average value (12.31 (±6.21) lg/L) in the inner bay was higher than those in the outer bay and bloom areas. MAA concentrations also demonstrated higher values (10.75 (±5.01) lg/L) in bloom areas than the outer bay due to high phytoplankton biomass. As a result of MAA analysis of the in situ surface waters in Arctic Kongsfjorden, five different types of MAAs (shinorine (SH), palythine (PA), mycosporine–glycine (MG), porphyra-334 (PR), and asterina-330 (AS)) were found (Fig. 4). The MAA composition demonstrated different trends in the bloom area and others sampling locations. Porphyra-334, in particular, was found in very high proportions in the bloom area, with an average concentration of 5.07

(±3.35) lg/L. Porphyra-334 proportions were particularly high at stations B05 (56.1%), B06 (64%), and B08 (42.3%), which were located in water surrounding the bloom area (Fig. 5). Phytoplankton species (Thalassiosira sp. and Phaeocystis sp.) and MAAs composition were compared in the study region. The relative abundance of Thalassiosira sp. was closely correlated with shinorine and porphyra-334 (Table 4). These data corresponded with the phytoplankton community structure. The relative abundances of Thalassiosira sp. and Phaeocystis sp. in surface waters of the bloom area and the inner bay impacted the MAA composition (Fig. 7). A relatively high proportion of MG was recorded in the inner (5.05 (±3.03) lg/L) and outer bays (2.82 (±1.12) lg/L) compared with that in the bloom area. Palythine was the second dominant MAA compound in the inner (2.4 (±1.22) lg/L) and outer bays (1.3 (±0.74) lg/L) (Table 1). 3.2. Carbon fixation and MAA production rates following in situ incubation Carbon fixation and MAA production rates were determined using PC bottles exposed to PA and quartz bottles that transmit natural PAB in offshore Kongsfjorden waters (St. B09) and in inner Kongsfjorden Bay (St. T05), where Phaeocystis sp. is known to have a large presence. The carbon fixation rates at sampling station B09 were 24.12 (±4.25) lg C/L/h during a 72-h exposure to PA and 22.23 (±10.22) lg C/L/h during a 72-h exposure to PAB (Table 2). However, the carbon fixation rate for a 72-h in situ incubation at the Phaeocystis sp.-dominated station (T05) indicated that PABexposure (31.8 (±2.6) lg C/L/h) was significantly inhibited (p < 0.01) compared to PA-exposure (41.5 (±2.5) lg C/L/h) (Table 2). The production rates of individual MAAs identified through 13C labeling were different depending on sampling station (Fig. 6). At station B09, the production rates of SH, PR, and PA were lower when exposed to PAB than when exposed to PA (Fig. 6a). Porphyra-334 demonstrated the highest production rate with a value of 0.16

Table 1 Distribution of temperature, chl a, and MAAs concentrations in the surface seawater, at sampling stations, outer and inner Kongsfjorden Bay. Station

Longitude

Latitude

Temperature (°C)

Chl a (lg/L)

Total MAAs (lg/L)

SH (lg/L)

PA (lg/L)

PR (lg/L)

MG (lg/L)

AS (lg/L)

Inner Bay K01 K02 K03 K04 T01 T02 T03 T04 T05 A01 A02 A03 Average

11.97 12.05 12.11 12.14 11.75 11.93 11.96 12.11 12.06 12.11 12.00 11.75

78.93 78.92 78.91 78.91 78.96 79.00 78.98 78.95 78.93 78.93 78.95 79.00

0.29 0.22 0.57 0.68 0.04 1.33 0.77 0.95 0.86 – – 0.09

0.57 0.47 0.36 0.36 0.51 0.67 0.83 0.29 0.24 0.42 0.3 0.15 0.43 (±0.19)

8.5 (±1.3) 12.7 (±5.1) 16.1 (±0.7) 12.7 (±1.2) 6.1 (±0.9) 13.4 (±2.7) 3.1 (±0.6) 19.6 (±3.2) 12.7 (±1.3) 25.5 (±1.4) 5.8 (±0.9) 11.6 (±1.2) 12.3 (±6.2)

2.2 2.9 3.3 2.7 0.9 3.3 0.7 3.1 2.0 5.0 1.0 1.9 2.4

(±0.4) (±1.4) (±0.3) (±0.3) (±0.1) (±0.7) (±0.2) (±0.7) (±0.2) (±0.4) (±0.3) (±0.6) (±1.2)

2.8 3.6 4.3 4.3 1.9 4.1 0.9 5.8 4.2 5.5 1.8 3.0 3.5

(±0.5) (±0.9) (±0.2) (±0.3) (±0.3) (±1.0) (±0.2) (±0.9) (±0.4) (±0.5) (±0.4) (±0.4) (±1.5)

0.7 (±0.3) 1.5 (±1.4) 1.5 (±0.1) 0.6 (±0.2) 0.3 (±0.1) 1.1 (±0.2) 0.6 (±0.2) n.d. n.d. n.d. 0.3 (±0.03) 0.5 (±0.2) 0.6 (±0.5)

2.9 (±0.6) 4.7 (±1.4) 6.4 (±0.2) 5.1 (±0.9) 2.7 (±0.6) 4.3 (±1.0) 0.8 (±0.1) 9.4 (±2.0) 5.0 (±0.5) 11.5 (±0.5) 2.1 (±0.4) 5.5 (±0.5) 5.1 (±3.0)

n.d. 0.02 (±0.01) 0.6 (±0.1) n.d. 0.3 (±0.03) 0.7 (±0.1) n.d. 1.3 (±0.3) 1.5 (±0.3) 3.5 (±0.3) 0.7 (±0.2) 0.7 (±0.4) 0.8 (±1.0)

Outer Bay A05 A06 A07 C01 C02 C04 B01 B02 Average

11.00 11.00 10.75 11.00 10.75 10.75 10.50 10.50

79.03 79.02 79.00 79.08 79.08 79.17 79.00 79.08

– 0.32 0.23 – 0.03 0.06 0.00 0.10

0.67 2.09 1.54 0.61 0.69 0.76 0.73 0.36 0.93 (±0.58)

9.7 6.8 7.7 9.3 3.9 4.5 4.1 9.3 6.9

2.0 1.5 0.9 2.6 0.7 0.6 0.6 1.6 1.3

(±0.6) (±0.2) (±0.02) (±0.7) (±0.2) (±0.2) (±0.3) (±0.03) (±0.7)

2.5 0.9 1.2 2.4 1.2 1.4 1.4 2.7 1.7

(±0.7) (±0.1) (±0.2) (±0.3) (±0.1) (±0.4) (±0.2) (±0.1) (±0.7)

0.7 1.5 1.0 0.7 0.3 0.3 0.3 0.7 0.7

(±0.2) (±0.4) (±0.2) (±0.03) (±0.04) (±0.1) (±0.2) (±0.1) (±0.4)

3.8 2.7 4.4 3.5 1.6 1.9 1.4 3.4 2.8

(±1.0) (±1.4) (±1.9) (±0.6) (±0.2) (±0.4) (±0.3) (±0.4) (±1.1)

0.7 0.3 0.3 0.2 0.2 0.4 0.4 0.9 0.4

(±0.1) (±0.1) (±0.03) (±0.1) (±0.1) (±0.1) (±0.04) (±0.01) (±0.2)

Bloom area B03 B05 B06 B08 B09 Average

10.50 10.25 10.25 10.00 10.00

79.17 79.08 79.17 79.08 79.17

0.20 0.26 0.22 0.25 0.54

1.13 6.8 5.36 3.58 2.63 3.90 (±2.23)

3.5 (±0.4) 17.3 (±1.3) 10.9 (±1.9) 9.5 (±1.2) 12.5 (±0.4) 10.8 (±5.0)

0.7 2.7 1.2 1.6 2.6 1.8

(±0.1) (±0.5) (±0.1) (±0.2) (±0.1) (±0.9)

0.8 1.9 1.2 1.9 2.6 1.7

(±0.1) (±0.4) (±0.04) (±0.1) (±0.03) (±0.7)

1.0 9.7 7.1 4.1 3.6 5.1

(±0.2) (±0.6) (±1.6) (±0.9) (±0.1) (±3.4)

0.9 2.0 1.0 1.6 3.3 1.8

(±0.1) (±0.2) (±0.1) (±0.03) (±0.3) (±0.9)

0.1 1.1 0.4 0.3 0.4 0.5

(±0.03) (±0.2) (±0.1) (±0.1) (±0.1) (±0.4)

(±2.6) (±2.0) (±2.2) (±1.1) (±0.3) (±1.1) (±0.8) (±0.5) (±2.5)

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100

Percentage (%)

80

60

40

20

0 A01 A02 A03 A05 A06 A07 B01 B02 B03 B05 B06 B08 B09 C01 C02 C04 K01 K02 K03 K04 T01 T02 T03 T04 T05

Fig. 5. Cumulative distribution of individual MAA compounds at all stations. Dark bar: shinorine, gray bar: palythine, dark gray bar: porphyra-334, light gray with slash: mycosporine–glycine, and dark gray with slash: asterina-330.

Table 2 Carbon fixation rate of in situ phytoplankton comparative analysis between treatments using Mann–Whitney test.

**

(lg C/L/h)

B09-incubation

T05-incubation

PA-treatment PAB-treatment

24.1 (±4.2) 22.2 (±10.2)

41.5 (±2.5) 31.8 (±2.6)**

Table 4 Correlation coefficient between individual MAAs production rates and phytoplankton species in this study region.

Thalassisosira sp. Phaeocystis sp.

p < 0.01

Shinorine

Palythine

Porphyra334

MG

Asterina330

.996** .098

.907 .342

.992** .562

.930 .282

.521 .480

MG: Mycosporine–glycine. Significance at p 6 0.01.

**

Table 3 Results of two-way ANOVA analysis on production rate, chl a specific production rate, and turnover rate of MAAs at station B09 and T05 during in situ incubation Station

Production rate Shinorine Palytine Porphyra-334 Mycosporine– glycine Asterina-330

Treatment

Station  treatment

F

P

F

P

F

P

123.112 48.654 4.035 181.578

.000⁄ .000⁄ .085 .000⁄

9.725 5.847 .385 10.453

.012⁄ .039⁄ .555 .012⁄

8.296 1.869 – 8.503

.018⁄ .205 – .019⁄

223.989

.000⁄

2.239

.173

3.045

.119

.000⁄ .000⁄ – .000⁄

.236 .013 .773 39.092

.638 .912 .413 .000⁄

.146 .257 – 37.064

.711 .625 – .000⁄

.000⁄

18.787

.002⁄

19.714

.002⁄

90.700 9.336 – 18.082

.000⁄ .014⁄ – .003⁄

11.522 8.197 3.276 3.266

.008⁄ .019⁄ .120 .108

7.309 1.470 – .316

.024⁄ .256 – .590

58.717

.000⁄

4.546

.066

7.359

.027

Chl a specific production rate Shinorine 106.280 Palytine 108.594 Porphyra-334 – Mycosporine– 218.312 glycine Asterina-330 180.311 Turnover rate Shinorine Palytine Porphyra-334 Mycosporine– glycine Asterina-330

The bold values indicates statistically significant of MAAs. * Statistically significant (p < 0.05).

(±0.09) ng C/L/h when exposed to PA and 0.09 (±0.08) ng C/L/h when exposed to PAB. The production rate of PA was 0.98

(±0.72) ng C/lg chl a/h following exposure to P and 0.47 (±0.39) ng C/lg chl a/h following exposure to PAB (Fig. 6b). The production rate and chl a production rate of PA were relatively high compared with those of the other MAAs following PA exposure. However, MAA turnover rates at B09 were slower following PA exposure than PAB exposure, with the exception of SH and PA. As a result, SH was determined to have the fastest production rate among the MAAs observed (Fig. 6c). The production rates of MAAs at T05 were found to be significantly higher than at station B09 following a 72 h incubation period. Production rates of individual MAAs at T05 were 10 times higher than those at B09 due to different dominant phytoplankton species. Individual MAAs production rates at station T05 demonstrated higher values following PA-exposure compared to those after PAB-exposure. The production rate of SH was 2.17 (±0.51) ng C/L/h, while the chl a-specific production rate of SH was 27.76 (±8.65) ng C/lg chl a/h following PA-exposure. On the other hand, the production rate of SH was 1.28 (±0.25) ng C/L/ h, and the chl a specific production rate was 25.55 (±4.97) ng C/ lg chl a/h following PAB-exposure (Fig. 6d and e). The turnover rates of individual MAAs exhibited somewhat similar patterns for production rates and chl a-specific production rates following PA and PAB-exposure at T05 (Fig. 6f). However, PA demonstrated significantly different turnover rates following PA and PAB-exposures. Looking at stations B09 and T05, SH had the highest production rate at T05, but PA had a relatively high production rate at B09 compared with those of all other MAAs.

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3.0

(a)

(d)

Production rate (pg C per L per hr)

Production rate (pg C per L per hr)

0.30 0.25 0.20 0.15 0.10 0.05

2.5 2.0 1.5 1.0 0.5

0.00

0.0

SH

PA

PO

MG

AS

PO

MG

AS

SH

PA

PO

MG

AS

PO

MG

AS

(e)

(b) Chl.a specific production rate (pg C per chl. a per hr)

Chl.a specific production rate (pg C per chl. a per hr)

PA

40

1.8 1.6

SH

1.4 1.2 1.0 0.8 0.6 0.4

30

20

10

0.2

0

0.0 SH

PA

PO

MG

AS

0.16

0.030

(c)

0.14

Turnover rate (per hr)

Turnover rate (per hr)

0.025 0.020 0.015 0.010 0.005 0.000

(f)

0.12 0.10 0.08 0.06

**

0.04 0.02

SH

PA

PO

MG

AS

0.00 SH

PA

Fig. 6. Production rates of individual MAAs in in situ phytoplankton incubation. (a) and (d): MAA production rate, (b) and (e): chl a-specific production rate of MAAs, (c) and (f): Turnover rates of individual MAAs, (a–c): station B09, (d–f): station T05, black bar: P-exposure, gray bar: PAB-exposure. Comparative analysis between P-exposure and PAB-exposure using Mann–Whitney test: ⁄⁄p < 0.01.

Production rates, chl a specific production rates, and turnover rates of individual MAAs indicated significant difference between both station B09 and T05 (p < 0.05), except for porphyra-334 due to no detection of porphyra-334 at T05. Likewise, shinorine, palytine, and mycosporine–glycine showed apparently different responses against UV-B through the treatment of light quality (PA and PAB) using two-way ANOVA. In particular, production rates of shinorine and mycosporine–glycine were affected by locations as well as UV-B radiation (Table 3), and also turnover rates of shinorine was significantly influenced by both locations and UV-B radiation (p = 0.024, p < 0.05).

4. Discussion 4.1. Comparison between individual MAA compositions and dominant phytoplankton species The composition of individual MAAs and dominant phytoplankton species may be a result of the biogeochemical processes occurring in the study area. The sea ice is reported to cover the inner bay of Kongsfjorden for 5–7 months per year, but sea ice does not form outside of the bay [38]. A 600 m-deep high temperature and hypersaline water of Norwegian Atlantic Current called West Spitsbergen

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Fig. 7. Distribution of chl a concentrations and total MAA concentrations as well as relative abundances of Thalassiosira sp. and Phaeocystis sp. in surface waters of Konsfjorden Bay and offshore.

Current (WSC) flows into the Arctic Ocean where the Svalbard archipelago is located, and Kongsfjorden is especially affected by the high temperature and hypersaline current flow that follows the west side of Spitsbergen island [38]. Slightly higher surface seawater temperatures were observed at station A05, located at the entrance of Kongsfjorden toward the outer bay waters (Table 1). Haptophytes, like Phaeocystis pouchetii, and diatoms, including Chaetoceros socialis and Thalassiosira nordenskioeldii, have been reported in high concentrations during the spring in Kongsfjorden [39]. In particular, P. pouchetii is a phytoplankton known to trigger spring blooms in the northern and Arctic waters [46,48]. Inner and central regions of Kongsfjorden Bay, which were covered with sea ice, were dominated by small diatoms or flagellates, whereas waters under the sea ice mainly have small phytoplankton such as flagellates as the dominating species [39,46,47]. The most dominant phytoplankton throughout the Kongsfjorden Bay was Phaeocystis sp., which was widely distributed at all sample stations. In addition to Phaeocystis sp., the nano-sized Thalassiosira sp. also had a dominant presence in the inner bay of Kongsfjorden, which may be influenced by the sea ice in the bay. This result corresponds with those of Kang et al. [37] and Wiktor [49]. Diatoms dominated the offshore waters of Kongsfjorden, which were affected by somewhat warm current, and were found to have low values of total MAAs despite of the high concentrations of chl a. In contrast, the inner of Kongsfjorden dominated by Phaeocystis sp. had low values of chl a but showed high concentrations of total MAAs (Fig. 7). The results of this study, as shown most evidently at stations B01 and A05, indicate remarkable differences in phytoplankton compositions, despite that fact that stations have relative proximity and similar chl a concentrations. Total MAA concentration at station A05, which was heavily populated with Phaeocystis sp., was nearly twice as high as that at station B01, which had a large concentration of diatoms (Thalassiosira sp.) (Fig. 7). At station T05, 96% of the Phaeocystis sp. in the phytoplankton community exhibited the lowest chl a concentrations of all the sample stations but the highest MAA concentrations due to Phaeocystis sp. This difference might be caused because Phaeocystis sp. is a species widely known to have strong UV absorption [13,33,50–52], and P. pouchetii in colony form have a strong absorption between 250 and 370 nm [50]. However, because the observed concentration of UV-absorbing compounds is too high to be intracellularly contained when P. pouchetii is in

the colony phase, most UV-absorbing compounds are considered to exist only in the extracellular colony matrix [33,50]. Little MAAs were found in a single Phaeocystis cell [50]. Phaeocystis is able to allocate its carbon reserves between the cell and the colonical mucilage [53]. Accordingly, some MAAs may be excreted into the colonical mucilage and within the colony, which would afford Phaeocystis an even greater protection than if they are solely located within the cell [52]. Consequently, inner Kongsfjorden demonstrated high MAA concentrations despite the low chl a concentrations. The abundance of PR in MAA compounds relatively higher at stations (B01, B03 and B09) in the offshore waters of Kongsfjorden compared to the other stations, and this result is corresponded to the previous reports that diatoms contain more SH and PR than other phytoplankton species (Fig. 5) [7,33,51,54,55]. Therefore, our results suggest that phytoplankton species composition should affect the spatial distribution of individual MAAs composition in Kongsfjorden. 4.2. Production rates of individual MAAs in the in situ phytoplankton community Seawater used for in situ incubation experiments was collected from bloom waters at stations B09 and T05. The waters from inner Kongsfjorden, dominated by Phaeocystis sp., were exposed for 72 h to examine changes in carbon fixation and MAA production rate caused by natural UV radiation (average of 13.2 W m2) following the addition of a 13C tracer. In this study, the community composition of phytoplankton species was determined by using microscopic analysis. Both Thalassiosira sp. and Phaeocystis sp. were predominant in Kongsfjorden Bay. Thalassiosira sp. occupied around 59% and Phaeocystis sp. contributed 40.9% to total phytoplankton cells at station B09. Also, Phaeocystis sp. occupied on average, 96.3% of total algal cell number at T05 station. So, the contribution of other phytoplankton species could be very minor to the overall production of MAAs. The production rates of individual MAAs determined the existence of a defense strategy against UV radiation in the in situ phytoplankton community. And the 13C assimilation experiments in combination with HPLC and EA-irMS were used to determine the production rate of individual MAAs through photosynthesis and to estimate the turnover rate of the particulate MAAs. The

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photoinduction of MAAs through labeling was clarified using the carbon stable isotope as a tracer. The reduced transmission (around 15%) of PAR may influence total C fixation rates as well as MAAs production rates, but it also might be in the range of saturated light intensity on account of high levels of solar irradiation during the in situ incubation experiments. The biosynthesis of MAAs is presumed to be occurring via the first part of shikimate pathway [56–58], but concluding evidences are lacking to a great extent [59]. It has been found that 3-dehydroquinate, which is formed during shikimate pathway, acts as a precursor for the synthesis of fungal mycosprines and MAAs via gadusols [26,34]. The primary MAA mycosprine-glycine thus synthesized by shikimate pathway is then transformed through chemical and/or biochemical conversions into secondary MAAs [56–58]. The synthesis of MAAs occurs in bacteria, cyanobacteria, phytoplankton and macroalgae but not in animals, because they lack the shikimate pathway [59]. These are some different mechanisms for effect of UV radiation on two phytoplankton species (diatoms and Phaeocystis sp.). Karentz [23] reported that the ratios of diatom surface area to volume were decreased to reduce the cellular damage against UV radiation. Diatoms have also been observed to enlarge their cell volume spontaneously without dividing when exposed to UV radiation [23]. On the other hand, UV radiation should be attenuated on the Phaeocysis cells according as light must be transmitted through the Phaeocysis colony and the fluid-filled space in its cgelatinous matrix, because Phaeocysis cells are generally embedded in a gelatinous matrix in natural ocean environments and have the excreted MAAs into the fluid-filled space or mucilage [52]. MAAs play a role as a passive sunscreen, exhibiting that MAAs are located in the cytoplasm [60] and are not coupled to the photosystems. Because MAAs are not involved to the photosynthetic antenna, there is no physical mechanism that would allow them to share excited electrons with the photosynthetic unit [52]. The energy absorbed by MAAs is not transferred to chlorophyll a and does not participate in photosynthesis [52]. So, carbon fixation through photosynthesis is not directly associated with MAAs accumulation in a cell. There was evidence of the photo-inhibition of UV radiation based on the total carbon fixation rates of phytoplankton incubated for 72 h. Although the relative abundance ratios of Phaeocystis sp. and diatoms in the in situ phytoplankton community were different at stations B09 (Phaeocystis sp.: 40.9%) and T05 (Phaeocystis sp.: 96.3%), the carbon fixation rates at both stations were affected by UV radiation. For the in situ incubation experiments conducted in Kongsfjorden, the UVR inhibition was found to be lower in Thalassiosira sp. (7.9% (B09)) than in Phaeocystis sp exposed to PAB for 72 h (23.4% (T05)). Previous reports indicate that phytoplankton carbon fixation in Antarctic and temperate waters is inhibited up to 25–50% by UV radiation [61,62], and that phytoplankton primary productivity in the Antarctic Ocean is inhibited by 6.4–60% [11,14,51,63,64]. The mechanisms of photoinhibition by UV radiation potentially direct and indirect effects on the major CO2 fixing enzyme ribulose-1,5bisphosphate carboxylase/oxygenase (Rubisco) [65]. Additionally, direct effects on cellular DNA can affect cell division and survival in diatoms [23]. Rubisco is sensitive to H2O2, which is produced in large quantities within the chloroplast during photooxidative stress [66] Moreover, as adenosine triphosphate (ATP) and NADPH2 are consumed during carbon metabolism, reduced levels of ATP could decrease carbon fixation in phytoplankton depending on the length of exposure to UV-B radiation [67]. The PSII reaction center is also known to be a target for damage by UV-B radiation in isolated thylakoids [68,69]. At station T05, where Phaeocystis sp. was predominant (96.3%) despite the active production rate of MAAs compared to those at

11

other stations (Fig. 7), the photo-inhibition of carbon fixation as a result of UV radiation was evident (Table 2). Phaeocystis species are reportedly very sensitive to UV radiation in artificial and ambient light compared to Antarctic diatom species [11,33,50,51]. However, a previous report suggests that Phaeocystis sp. demonstrates significant growth-inhibition by UV radiation compared to the diatom C. socialis [11]. Although the carbon fixation rate was photo-inhibited by UV radiation, MAA production rates increased in both PA and PAB exposure conditions in this study. This result was also obtained in a similar study [70]. Phytoplankton communities exposed to UV radiation require a combination of various factors in cell metabolism, including effective protection or recovery of the cell and an avoidance response to maintain survival and viability [33]. Exposure to UV radiation results in a positive correlation between carbon fixation rate and MAA concentration in the natural phytoplankton community, which suggests effective photoprotection [7,71–74]. Several studies of natural populations have suggested that MAA induction is related to irradiance [71,75]. Vernet et al. [13] observed that UV absorption in Antarctic phytoplankton, presumably due to MAAs, was higher in surface waters and decreased at depth. However, other studies have not observed protective effects of MAAs that prevent photosynthetic inhibition by UV radiation in Antarctic phytoplankton or freshwater microalgae [27,76]. The relationship between MAA synthesis and PAR intensity has been shown for dinoflagellate cultures [74,77]. Additionally, several studies have shown significant amounts of in vivo absorption under PAR limitation [13,60] and increased absorption with increased exposure to UV [52]. Some study reported that the production of MAAs was under the control of UVB exposure so that not only could induction of synthesis be elicited by UVB, but also the steady-state MAA contents in the cells reflected the UVB dose in many cyanobacteria [58]. Induction of MAA synthesis is also osmotic stresses which would preferentially activate the synthesis of mycosporine–glycine [58]. In various marine algal species, the synthesis of MAAs has been demonstrated to be induced either by UVB, UVA or PAR (400–700 nm). Especially, diatom produced the MAAs content in the cell from blue light (450–495 nm) and UV-A radiation [23], Phaeocystis sp. also excited the blue light and UV radiation due to response the biosynthesis of MAAs by wavelength [23,29]. Therefore, there are not significantly different between PA and PAB using two-way ANOVA (p > 0.05) in our results (Table 3). The composition of phytoplankton community in Kongsfjorden significantly affected the production rate of individual MAAs at both stations (Table 3). These results suggest that the different production rates of individual MAA compounds between station B09 and T05 should be caused by different MAAs synthesis of in situ phytoplankton assemblage (mainly Thalassiosira sp. vs. Phaeocystis sp.) against UV radiation (Fig. 7). Many biochemical UV-absorbing compounds (e.g., melanin, flavonoids, and anthocyanin) are reported to play an important role in the protection of cells from UV radiation [78–81]. Carotenoid pigment, produced by the xanthophylls cycle, functions as an antioxidant and disperses light energy in order to prevent intracellular photoinhibition from occurring in phytoplankton exposed to excessive light [26,82]. Several carotenoids, especially diadinoxanthin and diatoxanthin, function as important photoprotective pigments in phytoplankton such as diatoms, dinoflagellates and prymnesiophytes [83–86]. In addition, the intracellular accumulation of DMSP in phytoplankton may be accelerated by strong light and UVR nutrient limitation, which reflects the function of DMSP as an antioxidant to relieve photo-oxidative stress [87,88]. A recent experiment also showed that in several types of phytoplankton cultured indoors, the intracellular accumulation of DMSP was influenced by the UVR intensity [89]. Therefore,

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MAAs alone was not sufficient to completely protect the photosynthetic organs of phytoplankton cells from UV radiation, suggesting that MAAs may work with other UV-absorbing compounds to protect cells against exposure to UV radiation. The phytoplankton community in in situ waters at station B09 demonstrated an abundance of diatoms (Thalassiosira sp., 59%) over Phaeocystis sp. For diatoms, differences in MAA composition can vary depending on the morphological characteristics of the diatom [7,23]. The MAA content of centric diatoms is produced within 2 days through induction by UVA and shorter wavelength visible light (350–470 nm), and the concentration is maintained for at least a week [23]. For Phaeocystis sp., MAA concentrations increased more readily under UV-exposure conditions compared with PAR conditions; however, centric diatoms (Porosira glacialis (Grunow) Jorgensen) maintained MAA concentrations at a constant level regardless of the light conditions [70]. According to these results, there was no large difference in MAA production rates between UV-B blocked conditions and UV-B exposure conditions at station B09 because of the MAA synthesis mechanism of the dominating centric diatom species. The bioaccumulation of MAAs in dinoflagellates Gyrodinium dorsum Kofoid & Swezy and Phaeocystis antarctica Karsten is strongly induced by mediumlength UV radiation as well as PAR [23,89]. In P. antarctica in particular, the concentration of MAAs can vary depending on the strength of PAR [52]. In other words, the amount or the composition of MAA compounds varies according to the PAR and the UV wavelength. This variation in response to light occurs because each phytoplankton species has a different mechanism of MAA synthesis [23]. Stations B09 and T05 demonstrated clear differences in phytoplankton community structure and differences in individual MAA production rate (Fig. 6). Station B09, which was location in bloom waters, demonstrated low MAA concentrations and production rates despite a significantly high chl a concentration due to the dominance of diatoms in the water and the fact that the outside frustules of the diatom cells block more than 30% of UV radiation to protect the cells from harmful effects [23,51]. At station T05, however, greater than 96% of the species are Phaeocystis sp., and MAA production rates are 10 times higher than those at station B09 despite a low chl a concentration. This indicates a biochemical response elicited in Phaeocystis sp. to improve survival. These findings are supported by similar results from P. pouchetii cultures exposed to UV in a laboratory [50,64]. High UV absorption in Phaeocystis has been demonstrated in cultures and natural populations [13,33,46,48]. A lot of previous researches demonstrate that the MAA content and composition of UV-exposed phytoplankton cells vary substantially for each species depending on the light quality, intensity, and exposure time [7,23,33,54]. Additionally, most of these results were determined using cumulative photo-induction data yielded by observing the concentrations of UV-absorbing compounds in a natural phytoplankton community [90] or through UV exposure experiments in a laboratory. However, this study provides valuable information on MAAs as UV-absorbing compounds and indicates that the in situ MAA synthesis of natural phytoplankton is more reliant on phytoplankton community structure than light quality. The in situ production rates of UVabsorbing compounds like MAAs, which were determined using 13 C tracer and High Performance Liquid Chromatography (HPLC) combined with isotope ratio mass spectrometry (irMS), can aid in the understanding of the biochemical response and physiological state of the natural phytoplankton community against varying UV stressors. This measurement methodology also has the advantage of tracking real-time changes in newly photosynthesized MAAs as UV-absorbing compounds in the natural phytoplankton community.

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