Journal of Experimental Marine Biology and Ecology 429 (2012) 7–14

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Journal of Experimental Marine Biology and Ecology

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Effect of temperature on the photosynthetic efficiency and morphotype ofPhaeocystis antarctica

Fraser Kennedy, Andrew McMinn ⁎, Andrew MartinInstitute of Marine and Antarctic Studies, University of Tasmania, Private Bag 129, Hobart, Tasmania 7001, Australia

⁎ Corresponding author. Tel.: +61 3 6226 2980; fax:E-mail address: Andrew.McMinn@utas.edu.au (A. M

0022-0981/$ – see front matter © 2012 Elsevier B.V. Alldoi:10.1016/j.jembe.2012.06.016

a b s t r a c t

a r t i c l e i n f o

Article history:Received 2 May 2012Received in revised form 17 June 2012Accepted 18 June 2012Available online xxxx

Keywords:MesocosmPhaeocystis antarcticaSea iceTemperature

One of the only non-diatom species to dominate sea-ice assemblages is the haptophyte Phaeocystis antarctica.Here, the photosynthetic efficiency and morphotype expression of P. antarctica in response to freezing andmelting in an artificial sea-ice habitat is investigated. Maximum quantum yield of photosystem II (Fv/Fm)was significantly different with respect to both light (light and dark, Two-way ANOVA, pb0.001) anddepth within the ice (Two-way ANOVA, pb0.001). There was a decline in maximum quantum yield (Fv/Fm) in cells at each level within the ice, but the decline was greater in the coldest part of the ice (i.e. closeto the surface) than at the ice/water interface. Following the initiation of a melt cycle, Fv/Fm increased inboth treatments, from 0.48±0.05 to 0.57±0.05 on day 10, and 0.38±0.06 to 0.44±0.07 on day 10, in thelight and dark treatments respectively. The ice matrix induced solitary cell formation while melting inducedcolony formation. This change in morphology is not thought to reflect either temperature or nutrients but thephysical presence of ice acting as a trigger for morphological change.This study utilised a novel ice tank technology to replicate sea-ice habitat and document the response of P.antarctica to freeze/thaw dynamics.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

Physicochemical variability in the Southern Ocean influences the lifestrategies of many micro-organisms. Low temperature and extreme sea-sonal variation in photo-period requires significant physiological adapta-tions for survival (Kirst and Wiencke, 1995; McMinn et al., 2003; Reeveset al., 2011; Thomas and Dieckmann, 2002). Physiological and morpho-logical flexibility in microalgae is a response to both competition and en-vironmental pressures, and this has been akey to their survival not only inpolar environments but also in most aquatic systems (Arrigo et al., 1997;Thomas and Dieckmann, 2002).

However, the most significant environmental change in the South-ern Ocean is the annual formation of sea-ice around the Antarctic conti-nent. This ephemeral body of ice not only provides an extensive habitatfor many organisms but is also a significant climate regulator. Whenseawater freezes, it forms a semi-solid matrix with a complex systemof brine channels and pores (Thomas and Dieckmann, 2002). Microbesinitially scavenged from the water column are concentrated within thisice matrix during ice formation (Martin et al., 2011; Mock and Thomas,2005). The sea-ice habitat experiences extreme variation in tempera-ture, salinity, nutrients and light and is increasingly being recognizedas a proxy for extra-terrestrial systems (Chyba and Phillips, 2001). Forexample, salinity can fluctuate from 0 to 215 ppt (Mock and Thomas,2005), while the under-ice irradiance, even during summer can be

+61 3 6226 2973.cMinn).

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reduced to less than 5 μmol photons m−2 s−1 compared to a surface ir-radiance of approximately 1700 μmol photons m−2 s−1 (McMinn etal., 1999). While the range of microbes initially scavenged is typicallydiverse, themicroalgal fraction of the sea icemicrobial community is ul-timately usually dominated by a small number of pennate diatom spe-cies (Arrigo et al., 1997; Peloquin and Smith, 2007; Thomas andDieckmann, 2002). Within fast-ice, these cells are typically concentrat-ed in the bottom or skeletal layer of the ice but sub-ice communities canalso be present depending on the presence of under-ice currents andproximity to ice shelves (McMinn, 2010; Mock and Thomas, 2005). Al-though sea ice algae are present in the interior and near-surface regionsof the ice, the steep environmental gradients in temperature, salinityand nutrients within the ice fabric limit the ability of many species tosurvive (Robinson et al., 1997).

One of the few non-diatom species known to dominate sea-ice as-semblages, particularly in the Arctic, is the haptophyte, Phaeocystisantarctica, which has been reported at cell concentrations of up to5×107 cells l−1 (Fryxell and Kendrick, 1988; Garrison et al., 1987).P. antarctica is arguably one of the most abundant micro-algal speciesin Antarctic waters (Davidson, 1996; Marchant and Scott, 1993) andis commonly observed during the austral spring, where it oftenforms dense post-melt blooms, particularly in coastal areas and theRoss Sea (Moisan and Mitchell, 1999; Rousseau et al., 2007). Cell con-centrations of P. antarctica during phytoplankton blooms have beenreported to reach 6×107 cells l−1 (Davidson and Marchant, 1992)and can account for 38% of the primary production in some areas ofthe Southern Ocean (Palmisano et al., 1986).

8 F. Kennedy et al. / Journal of Experimental Marine Biology and Ecology 429 (2012) 7–14

P. antarctica has two main life cycle forms; a motile, bi-flagellateform and a colonial form. The motile, flagellate morphotype is relative-ly small (3–5 μm), bearing two greenish-brown chloroplasts, two equallength flagella, a short bulbous haptonema and a periplast commonlybearing two layers of scales (Rousseau et al., 2007). Flagellate cellscommonly exude threads from two membrane bound vesicles, whichform five armed ‘star’ arrays (Scott and Marchant, 2005). It has beenproposed that the threads are released upon vesicle rupture and thenstraightened to form the diagnostic pentagons or ‘stars’ (Davidson,1996; Pienaar and Cooper, 1991). However, the exact function ofthese threads remains unknown.

P. antarctica is most commonly observed in its colonial form, whereit is often the main constituent of blooms in Antarctic waters. Cellswithin the colonial matrix lack scales, flagella and haptonema and arerelatively large, ranging from 5 to 7.5 μm (Davidson, 1996; Rousseauet al., 2007; Scott and Marchant, 2005). These cells are contained with-in a membrane, which is solute permeable and composed mainly ofphoto-assimilated carbon that has been secreted from vesicles throughthe plasmalemma (Hamm et al., 1999). Benefits of the colonial mor-phology include protection from grazers (Verity and Medlin, 2003), re-duced bacterial contamination (Davidson and Marchant, 1987; Shieldsand Smith, 2009), enhanced UVB protection (Davidson, 1996) and theincreased ability to share photo-assimilated products. Despite the ex-tensive research that has been conducted on Phaeocystis, its physiolog-ical and morphological response to biotic and abiotic environmentalfactors is mostly lacking and little research has been conducted onthe photosynthetic and morphological response to freeze/thaw dy-namics. Even less research has been undertaken on the examinationof the photosynthetic physiology of P. antarctica within sea-ice ecosys-tems (Tang et al., 2009) and the life history strategy of P. antarctica dur-ing winter remains unknown (Tang et al., 2009). Here, we investigatethe photosynthetic efficiency and morphotype response of P. antarcticato the freeze/thaw processes using a novel artificial sea-ice habitat.

2. Materials and methods

2.1. Culture material

Cultures of P. antarcticawere obtained from the Australian AntarcticDivision (Kingston, Tasmania). Colonial and flagellate life cycle stageswere both represented in the culture material, as determined by lightmicroscopy and the descriptions of P. antarctica found in Scott andMarchant (2005). In the laboratory, cultures of P. antarctica weregrown semi-continuously in f10 medium (Guillard and Ryther, 1962)at 2 °C±2 °C, under continuous cool white fluorescence light(60 μmol photon m−2 s−1). Light intensity was measured with a labo-ratory quantum scalar irradiance meter (Biospherical Instruments QSL-100).

2.2. Sampling

Light and dark treatment experiments were performed in both anice tank and a water bath. The water bath experiments were designedto expose the cells to the same temperature and light conditions asthe ice tank but without the presence of ice.

2.2.1. Ice tankThe ice tank was engineered to replicate Antarctic pack ice condi-

tions in the laboratory. The main chamber, which had a capacity of70 l and a diameter of 80 cm, was kept inside a chest freezer in whichtemperature could be accurately controlled from −5 °C to −30 °C.The sides and bottom of the chamber were insulated so cooling andice formation would only occur from the top. In the experiments de-scribed herein the temperature in the freezer was set to −15 °C. Sea-ice formation was aided by four variable-flow submersible pumps(Aqua Nova 400 —240 V) and a series of heaters that were initiated

depending on the required thickness of ice. An additional external sen-sor determined ambient temperature within the freezer environmentand acted in combination with a central sensor to enable/disablepumps and/or heat. The pumps ensured that the under ice water wasconstantly well mixed. A compensation tank was fitted to the outsideof the main tank and connected via a heated pipe to compensate forthe water displacement during sea ice formation. White light was pro-vided from above by an externally operated cool white LED light source,with a maximum output of 60 μmol photons m−2 s−1. Irradiance wasreduced to 28 μmol photon m−2 s−1 under 6 cm of ice (standard ex-perimental thickness).

Prior to each experimental run, the ice tank was acid washed with10%HCl and rinsedwithMilli-Qwater tominimise bacterial contamina-tion. Clean seawater was collected from Trumpter Bay, Bruny Island,Tasmania, Australia, pressure filtered through a 0.20 μm filter andstored for b24 h in an acid washed 70 l Nalgene carboy. Seawater salin-ity was determined before each experiment and adjusted (if necessary)to a value of 37 parts per thousand (ppt). Prior to inoculation, 66 l ofpre-filtered seawater with additional f10 nutrient (Guillard andRyther, 1962)was placed into the ice-tank and chilled to−1.5 °C beforethe P. antarctica culture was added at a final concentration of approxi-mately 1000 cells ml−1. To reduce the potential shock-inducedmortal-ity, cells were acclimated over a period of two days to the ice tanktemperature (−1.5 °C) and light conditions (30 μmol photon m−2 s−1)by using a Wisd WiseCircu WCB-12 waterbath (Witeg Germany). Tem-peraturewithin the icematrixwas recorded and loggedwith two iButtonDS1922L temperature data-loggers (Maxim designs, USA), with an accu-racy of ±0.05 °C. Loggers were placed within 2 cm of the top and bottomice surfaces during each experimental run.

The ice tank was set at a temperature of −15 °C for ice formation.Once the ice had reached its maximum thickness of 6 cm (after 48 h),daily ice and water sampling commenced and continued for10 daysfor each experiment. A maximum of eight ice cores were collectedfrom the tank daily using a manual hollow ice-screw (diameter1 cm): six were used for PAM fluorometry and epifluorescent micros-copy and two for chlorophyll-a analysis. Each core was immediatelydivided into three sections according to ice core depth, i.e. surface2 cm, middle 2 cm, bottom 2 cm; a further sample was taken fromthe underlying water. The 5 ml water sample was collected by syringefrom each ice hole following core extraction. Each ice subsection wascarefully placed into a pre-chilled Falcon test tube and placed on-icein the dark for no longer than 30 min. After a period of five days theice tank was placed into a ‘melt cycle’, which induced the pumpsand heat plate to constantly operate until the desired temperatureof −1.5 °C was reached in the water column. During the ‘melt cycle’the ambient freezer temperature was increased to−5 °C by manuallyadjusting the thermostat. Eight 15 ml water samples were collecteddaily for 5 days by sterile pipetting during the melt cycle and placedinto a pre-chilled Falcon test tube and placed on-ice in the dark forno longer than 30 min. The ice tank incubations were run fourtimes, twice in the dark and twice in the light.

2.2.2. Water bathA series of control experiments, using a Wisd WiseCircu WCB-12

(Witeg Germany) circulating water-bath with a 1:2 water:solvent(methanol) ratio was used to provide sub-zero temperatures and todifferentiate the effects of temperature from the effects of salinity.40 ml of pre-filtered (0.2 μm) seawater amended with f10 nutrient(Guillard and Ryther, 1962) was added to optically clear tissue cul-ture flasks and chilled to treatment temperature before cell inocula-tion. P. antarctica cultures were added to sample flasks until adesired concentration of approximately 1000 cells ml−1 was reached.Cultures were then acclimated for a period of two days. Two temper-ature treatments were applied: 0 °C±0.1 °C and −2 °C±0.1 °C, withcultures exposed to either light or dark. Dark treatment flasks werewrapped in aluminium foil to achieve total darkness, while light

Fig. 1. Observed temperature within the ice over time, lines represent different treat-ments (light and dark) and depths where temperature was recorded. Differentiationbetween the ice and melt cycles is represented by the dotted line.

9F. Kennedy et al. / Journal of Experimental Marine Biology and Ecology 429 (2012) 7–14

treatment flasks were exposed to 28 μmol photon m−2 s−1. Over aperiod of nine days, triplicate light and dark treatment flasks were ,sampled daily. From each sample, 10 ml was extracted for epi-fluorescencemicroscopy andpreserved in gluteraldyhyde (5%final con-centration), 15 ml for PAM fluorometry and 20ml for chlorophyll-aanalysis.

2.3. Pulse amplitude modulation fluorometry

Chlorophyll-a fluorescence of P. antarctica was measured using apulse-amplitude-modulated fluorometer (Water-PAM,Walz, Effeltrich,Germany)with an internal actinic light source centred on 660 nm. Sam-pleswere dark-acclimated for 30 min prior tomeasurement. Rapid lightcurves (RLC) were obtained under software control (WinControl,Walz). Maximum quantum yield in the dark-acclimated state, Fv/Fm,was obtained from the first value of the RLC when the irradiance was0 μmol photons m−2 s−1. Samples were exposed to a weak measuringlight (b1 μmol photons m−2 s−1), which induces fluorescence withoutstimulating photosynthesis and allows minimal fluorescence (F0 in thedark, F in the light). A second light source, a saturating pulse(>3000 μmol photons m−2 s−1 for 0.8 s), was used to close all reactioncentres and measure maximum fluorescence (Fm in the dark Fm′ in thelight). RLCs were comprised of a series of eight saturation pulsesfollowed by exposure to a sequence of increasing actinic light of 10 s du-ration (Ralph and Gademann, 2005;White and Critchley, 1999). The ef-fective quantum yield,ΔF/Fm′, was obtained after each exposure to eachactinic light level, which here was 31, 47, 71, 105, 162, 242, 346 and483 μmol photons m−2 s−1. The relative photosynthetic electrontransport rate (rETR) was calculated as the product of the effectivequantum yield of PSII and quantum flux density of photosyntheticallyactive radiation (PAR) (Genty et al., 1989). The rETR data generatedby the rapid light curves were fitted to the exponential function ofPlatt et al. (1980) using a multiple non-linear regression. In this caserETR was substituted for photosynthesis (P) in their P vs I relationshipand the photosynthetic efficiency (α) is subsequently referred to asthe initial slope of the rETR vs E curve.

rETR ¼ rETRmax � 1� exp �αEd=rETRmaxð Þ½ � exp �βEd=rETRmaxð Þ

As photoinhibition did not occur in the light curves, the functionreduces to:

rETR ¼ rETRmax 1� exp �αEd=rETRmaxð �:½

Gain settings on the PAM were consistently between 5 and 10.

2.4. Microscopy

Culture samples for use in epifluorescencemicroscopywere extractedfrom the ice matrix as described above. Melted ice core samples (5 ml)were immediately preserved in glutaraldehyde (5% final concentration)and stored in amber glass bottles in the dark at 2 °C. Prior to enumeration,sampleswere filtered underminimal pressure onto black 0.8 μmpolycar-bonate filters and stained with acridine orange (1% final concentration)for two minutes. The filters were immediately mounted onto glass slidesand visualized using a Zeiss Axioskop 2 fluorescence microscope. A blueexcitation wavelength of 460 nm was used to examine the morphologyof P. antarctica and undertake the cell counts. A total of twenty randomfields of viewwere used to count cell morphotype using a 100× objectiveunder oil immersion. Colonial and solitary cellswere classified by cell size,presence/absence of flagella and whether cells were embedded in amucus matrix.

2.5. Statistics

All analyses were performed using the statistical package R (version2.12.2). Two-way analysis of variance (ANOVA) was selected to test forsignificant differences within treatments, providing that the data metthe required assumptions. If heteroscedasticity was present, the datawas appropriately transformed. SigmaPlot 11.0.0.77 (Systat Inc) wasused for all graphical applications. Unless otherwise stated, all errorbars represent the standard error of the mean.

3. Results

3.1. Ice tank

During each experiment the temperature within the ice increasedwith depth, with the highest temperatures at the ice-water interfaceand the lowest at the ice surface (Fig. 1). Cultures of P. antarctica weresuccessfully incorporated into the ice matrix, and the majority of thecells were concentrated within the bottom 2 cm of ice (Table 1).When the abundance of cells incorporatedwithin the icewas comparedbetween experimental runs, no significant differences were observedwith respect to depth (Two-way ANOVA, light: p=0.521, dark,p=0.437).Maximumquantumyield of photosystem II (Fv/Fm)was sig-nificantly different with respect to both light (light and dark, Two-wayANOVA, pb0.001) and ice depth (Two-way ANOVA, pb0.001, Fig. 2). Inthe light treatment, cells that were closer to the ice surface displayedlower Fv/Fm than those present either in the middle or bottom sectionsof the ice, decreasing from a mean of 0.395±0.022 on day 1 to a mini-mum of 0.141±0.058 on day 5. Cells located in the middle of the icealso displayed this trend, decreasing from 0.432±0.029 on day 1 to0.198±0.020 on day 5. Cells located in the bottom ice communityalso declined but not as rapidly; the decrease was from 0.446±0.012to 0.307±0.028 on day 5. Cells in the water column showed no signifi-cant reduction in Fv/Fm, being 0.520±0.027 on day 1 and 0.516±0.044on day 5 (Fig. 2(A)). While there was a significant difference betweenFv/Fm in the light and dark treatments, the declining trends in Fv/Fmwith depth in the dark were similar to those in the light (Fig. 2(B)).

In the dark treatment, the Fv/Fm of the surface ice community de-clined from 0.153±0.022 on day 1 to 0.032±0.010 on day 5. Cellswithin the middle ice depth showed a similar trend, decreasing from0.374±0.027 to 0.067±0.008. The bottom community also decreased,but only from 0.591±0.022 on day 1 to 0.255±0.048 on day 5. Fv/Fm ofcells in the underlying water declined from 0.552±0.051 on day 1 toonly 0.366±0.020 on day 5, a significantly greater decline than in thelight treatment (Fig. 2).

Table 1Total cell abundance of Phaeocystis antarctica in the ice and underlying water of the icetank by treatment (light, dark). Depth is in cm from top of ice, cell abundance is in cellsml−1.

Time Water Bottom Middle Top Melt

(4–6 cm) (2–4 cm) (0–2 cm)

Light treatmentDay 1 224±23 96±16 65±7 51±11 –

Day 2 172±6 160±9 78±11 51±3 –

Day 3 287±14 162±12 45±3 29±3 –

Day 4 319±11 118±13 40±2 8±3 –

Day 5 350±11 152±9 39±3 10±9 –

Day 6 – – – – 486±15Day 7 – – – – 500±17Day 8 – – – – 545±13Day 9 – – – – 522±16Day 10 – – – – 508±17

Dark treatmentDay 1 156±10 117±8 67±10 46±4 –

Day 2 161±10 119±10 76±4 43±3 –

Day 3 150±14 155±9 81±9 32±3 –

Day 4 157±7 102±9 99±3 16±3 –

Day 5 160±8 146±4 98±9 7±6 –

Day 6 – – – – 216±14Day 7 – – – – 226±8Day 8 – – – – 208±12Day 9 – – – – 210±10Day 10 – – – – 206±14

Fig. 2. Maximum quantum yield (Fv/Fm) of P. antarctica in the ice tank. Showing thelight (A) and the dark treatments (B). Each line represents a different depth with theice environment. Differentiation between the ice and melt cycles is represented bythe dotted line. After melting a combination of cells from under the ice and frommelted ice is measured.

10 F. Kennedy et al. / Journal of Experimental Marine Biology and Ecology 429 (2012) 7–14

Once themelt cycle had begun, it took approximately 12 h for the iceto melt; thus all ice tank measurements from day 6 onward are of acombined under ice water and melted ice community. Following themelt cycle, Fv/Fm increased in both treatments, from 0.478±0.05 to0.570±0.05 on day 10, and 0.375±0.06 to 0.442±0.07 on day 10, inthe light and dark treatments respectively.

rETRmax in the light treatment differed significantly according toice depth (one-way ANOVA, pb0.001); rETRmax of cells located inthe water column increased from 22.3±1.62 μmol electrons m−2 s−1

on day 1 to 29.5±1.26 μmol electrons m−2 s−1 on day 5 (Fig. 3). Inthe ice rRTR was highest near the ice surface (22.5±2.1 μmol electrons m−2 s−1 on day 1, decreasing to 3.88±0.66 μmol electrons m−2 s−1 on day 5), but decreased with depth(bottom, 11.3±0.90 μmol electrons m−2 s−1 on day 1 to 10.6±0.38 μmol electrons m−2 s−1 on day 5). The dark treatment, dis-played a similar response, although values of rETRmax were lower(Fig. 3).

The initial slope of the rETR vs E curve (approximately equivalentto photosynthetic efficiency, α) showed a similar pattern to Fv/Fm andrETRmax. Values decreased significantly over the five days of ice for-mation with the greatest decline at the top of the ice and the leastat the bottom. There was a significant increase in the under icewater values (Fig. 4).

Within the ice, solitary flagellate cells were the dominant mor-photype found in both the light and dark treatments (Fig. 5(A)).The colonial form was also present, but numbers decreased in eachtreatment. In contrast to the cells within the ice matrix, flagellates

Fig. 3. Relative electron transfer rate (rETRmax) of PSII in P. antarctica by depth andtreatment. A) light treatment, B) dark treatment. Differentiation between the ice andmelt cycles is represented by the dotted line. After melting a combination of cellsfrom under the ice and from melted ice is measured.

11F. Kennedy et al. / Journal of Experimental Marine Biology and Ecology 429 (2012) 7–14

(solitary cells) were significantly less abundant in the water column,ranging from 98±15 cells ml−1 on day 1 to 99±5 cells ml−1on day5, than colonial cells (which ranged from 125±12 cells ml−1 on day1 to 251±8 cells ml−1 on day 5 in the light treatment). In addition,colonial cells were also the dominant form present during the meltcycle in both treatments, although cell numbers of both forms weremuch lower in the dark and showed little sign of active growth(Table, Fig. 5(B)). Flagellate cell numbers remained relatively stablethroughout the experimental period for each treatment.

Towards the end of the ice period and throughout the melt periodthere was a heightened production of the diagnostic ‘five star-arrays’from flagellated cells (Fig. 6). These arrays appeared to interlock, cre-ating web-like structures, which enveloped other cells (Fig. 6(C)).This was only observed in ice-free water and not during the darktreatment. Production of arrays was not quantified.

3.2. Water-bath control experiments

Therewas a significant difference in Fv/Fm between the two temper-ature treatments (Two-way ANOVA, pb0.001) and between the twolight treatments (Two-way ANOVA, pb0.001). Themaximum quantumyield of cells maintained at −2°, both those incubated in the light anddark were consistently lower than the 0 °C treatment (Fig. 7). In gener-al, Fv/Fm values in all treatments increased with time during all experi-ments. There was no significant difference in rETRmax between the 0 °Cand−2 °C temperature treatments, although rETRmax was consistently

Fig. 4. Change in the initial slope of the rETR vs E curve (approximately equivalent tophotosynthetic efficiency, α) with depth and treatment over time. A) light treatment,B) dark treatment. Differentiation between the ice and melt cycles is represented bythe dotted line. After melting a combination of cells from under the ice and frommelted ice are measured.

Fig. 5. P. antarctica cellmorphotype as sampled in the ice tank during, A) light and B) darktreatments. Data shown in the total number of eachmorphotype sampled in the bottomofthe ice and those during icemelt. Note the increased number of colonial morphotype dur-ing ice melt. Differentiation between the ice and melt cycles is represented by the dottedline. After melting a combination of cells from under the ice and from melted ice ismeasured.

lower in both dark treatments. The initial slope of the rETR vs E curve(approximately equivalent to photosynthetic efficiency, α) was alsoconstantly lower in the dark, although there was no significant differ-ence between temperature treatments.

Colonial cells were observed to be the dominant morphotype inthe 0 °C light treatment (Fig. 8(A)), and cell abundance increasedfrom 143±2.85 cells ml−1 to 382±6.12 cells ml−1 on day 9. In thedark, both cell morphotypes displayed the same stable trend. At−2 °C there was no difference in the concentration of either mor-photype (Fig. 8(B)).

4. Discussion

Natural sea ice is characterised by steep physicochemical gradientsin temperature and salinity. Temperatures and salinities at the base ofsea ice are close to those of seawater, while temperatures at the surfaceand strongly influenced by ambient atmospheric conditions and salinityinversely co-varies with temperature (Tucker et al., 1992). Surface icetemperatures have been recorded to as lowas−20 °Cwith accompany-ing salinities of >200 psu; these conditions are well beyond what isnormally considered suitable for biological activity (Thomas andDieckmann, 2002). Ralph et al. (2005) have shown that sea ice algaeare able to remain photosynthetically active at temperatures down to−10 °C, but activity was drastically reduced and cells become photo-inhibited at increasingly lower levels of irradiance. The temperatureand salinity profiles of sea ice are therefore likely to play a critical rolein the distribution and physiological health of algal cells.

Fig. 6. P. antarctica stained with acridine orange as seen under epifluorescence throughout the melt cycle of the light treatment. Specifically showing the diagnostic ‘five arm stararrays’which were only observed during ice-free conditions during the light treatment. Figures (A), (B) are taken from replicate one; (C) and (D) from replicate two during the meltcycle of the light treatment. Note the lack of flagella in cells enveloped in the threads. (For interpretation of the references to colour in this figure legend, the reader is referred to theweb version of this article.)

12 F. Kennedy et al. / Journal of Experimental Marine Biology and Ecology 429 (2012) 7–14

P. antarctica is a dominant component of many Southern Oceanphytoplankton communities and contributes significantly to primaryproduction in the region (Arrigo et al., 2010; Schoemann et al.,2005). It is particularly characteristic of deeply mixed, lower light en-vironments, such as the Ross Sea, where it regularly outcompetes di-atoms (Alderkamp et al., 2012; Arrigo et al., 2010). Although sea icealgal communities are usually dominated by diatoms, Phaeocystisspp. is often present and can sometimes be dominant (Garrison etal., 1987; McMinn and Hegseth, 2004).

In the ice tank experiments described here cells of P. antarctica wereeffectively incorporated into brine pockets and channels within the icematrix during ice formation and were then successfully maintained

Fig. 7. Maximum quantum yield of P. antarctica cells in the water-bath experiment.Lines represent different light and temperature treatments. Note the higher yield ofcells in the 0 °C treatment.

through an ice growth and melt cycle. There was a decline in maximumquantum yield (Fv/Fm) in cells at each level within the ice, but the declinewas greatest in the coldest andmost saline part of the ice (i.e. close to thesurface). The interaction between temperature alone and maximumquantum yield and was further examined by observing the response ofcells in a water bath. In these control experiments, during which salinitywas held constant and nutrient levels remained high, Fv/Fm decreased atlower temperatures, a response thatwas observed irrespective of the irra-diance. Given that initial nutrient levels were high, incubation periodsshort, and the biomass levels low, it is not likely that the cells wouldhave become nutrient limited. This response, therefore, reflects the effectof lower temperature alone on cell photophysiology. Many studies havedocumented temperature dependent responses in a range of taxa includ-ing micro-algae (Davison, 1991; Epply, 1976), Phaeocystis (Verity et al.,1988b) and sea ice algae (Palmisano et al., 1986; Ralph et al., 2005,2007). However, there is a close relationship between temperature andsalinity in forming sea ice with the salinity in brine channels increasingas the temperature decreases (Tucker et al., 1992). The top section ofsea ice in the ice tank was exposed to temperatures of −10 °C to−15 °C which caused the salinity to increase to ~150–175 psu (Tuckeret al., 1992). Likewise, the salinity in the middle section was between~70 and 150 psu. Arrigo and Sullivan (1992) described the effect of thisco-variation on sea ice algae. Ralphet al. (2005) further showed that salin-ities over 65 psu had a serious effect of photophysiology and that changesin both these parameters would cause serious stress. Thus in this study itwas not possible to distinguish the separate effects of temperature and sa-linity at high levels in the ice, where temperature and salinity were ex-treme, as both were changing during ice formation. During natural iceformation, nutrients become concentrated both in brine pockets and im-mediately under the ice and are subsequently diluted during melting(Thomas andDieckmann, 2009). However as nutrient levelswere alreadyhigh in the ice tank experiments, it is unlikely that increased nutrient con-centrationswere responsible for the higher Fv/Fm values of the cells in the

Fig. 8. Total concentration of solitary and colonial cell morphotypes of P. antarctica overthe duration of the water-bath control experiment. Plots represent temperature treat-ments A) 0 °C and B) −2 °C, while bars are grouped by light treatment.

13F. Kennedy et al. / Journal of Experimental Marine Biology and Ecology 429 (2012) 7–14

lower sections of the ice. Interestingly, theminimum thermal tolerance ofP. antarctica has been previously reported to be approximately −2 °C,belowwhich growth rates and photosynthesis become severely impaired(Baumann et al., 1994). However, it is shown here that P. antarctica cantolerate and grow at temperatures down to −3 °C, although exposureto temperatures below −3 °C significantly inhibited photosyntheticperformance.

rETRmax also demonstrated a clear temperature relationship withdepth in the ice. rETRmax for cells from the surface of the ice (coldesttemperature) dropped dramatically from ~22 μmol electrons m−2 s−1

on day 1 to less than 4 μmol electrons m−2 s−1 on day 5. This compareswith an insignificant decline over time at the bottom of the ice over thesame period, from 11.3 μmol electrons m−2 s−1 to 10.6 μmol electro-ns m−2 s−1. The rETRmax of cells from the underlying water columnwas more than five times higher than those from the top of the ice. Aswith Fv/Fm, this change is likely to be related to a temperature threshold.While photosynthetic rates are known to be temperature dependent, itwas thought that the initial slope of the rETR vs E curve (approximatelyequivalent to photosynthetic efficiency, α) would be largely unaffectedby fluctuations in temperature (Falkowski and Raven, 1997). In the icetank, however, there was a systematic increase in this parameter withdepth in the ice and decrease with ice temperature; a response that wasthe same in both the light and dark. It is probable that this response isreflecting the impact of increasing salinity rather than temperature. Thephotosynthetic parameters of the melt water samples were relativelyhigh, and even higher than in the below-ice seawater, and this is likelyto be a result of increased temperature.

The experimental design did not examine the recovery of cellsfrom different depths within the ice or the underlying water, so it is

not known whether the cells exposed to the lower temperaturesalso recovered. However, final Fv/Fm values for the higher ice levelswere less than 0.1 and previous experimental work has shown thatcells with a Fv/Fm of b0.1 are unlikely to recover (Reeves et al.,2011). A gradual increase in Fv/Fm in the−2 °C water-bath treatment(both light and dark) over time illustrates some capacity of those cellsto acclimate to that temperature (Fig. 7). In contrast, cells exposed totemperatures below −3 °C in the ice-tank, i.e. in the middle and topsections of the ice, exhibited a decline in Fv/Fm and rETRmax valuesover time and there was no evidence for acclimation. A large andsignificant reduction in the Fv/Fm values of P. antarctica followingfreezing (0.6 to 0.28), was also observed by Tang et al. (2009) in asimilar mesocosm experiment. These authors did not measurephotosynthetic efficiency, but also suggested a temperature thresholdfor P. antarctica.

The original starting culture of the sea ice tank contained a mix-ture of both solitary and colonial cells. However, after ice formation,the proportion of solitary cells in the ice increased in both the lightand dark treatments. Conversely, in the water underneath the ice co-lonial cells grew rapidly (light treatment only) while solitary cellnumbers remained static and this trend continued following icemelt in the ice tank. This is consistent with the observation that for-mation of Phaeocystis spp. colonies occurs naturally during meltingof the sea ice in spring (Smith et al., 2003; Wassmann et al., 2005).Arrigo et al. (2010) reported that colonial forms exhibited highermaximum photosynthetic and growth rates than solitary cells in theearly stages of bloom events and were able to capitalize with respectto growth during a period of increased nutrient availability and levelsof irradiance. Change from a solitary to a colonial form may also be aresponse to minimise photo-inhibition caused by the sudden increasein irradiance during ice melt (Palmisano et al., 1986), to aid retentionin the subsequent mixed layer by reducing settling rates, or by pro-viding increased protection against predation (Verity et al., 2007).In this study, the ice matrix itself appears to induce solitary cell for-mation while melting induces colony formation. This change in mor-phology is not thought to reflect either temperature or nutrients, as achange in morphotype was not observed during the water bath ex-periments. It is also possible that the temperature range used in thewater bath was insufficient to initiate the morphological change andexperiments with a larger temperature range would be needed forconfirmation. Tang et al. (2009) reported an increased abundance ofthe solitary cell morphotype following ice formation. They also de-scribed a reduction in the size and physical dislodgement of colonialcells from within the colony membrane during freezing. Reduced col-ony size was not observed in the current study, although a decline inthe abundance of colonial cells was documented. Verity et al. (2007)also suggested that the solitary form of Phaeocystis may play an im-portant role as an overwintering strategy; i.e. a senescent statefollowed by attachment to a substrate. A morphological response byP. antarctica to environmental change has been reported for micro-nutrients (Alderkamp et al., 2012; DiTullio et al., 2007), grazing pres-sure (Verity et al., 1988a), irradiance (Arrigo et al., 2010) and alsotemperature (Tang et al., 2009). Here, we show that the physicalpresence of ice itself may also act as a trigger for morphologicalchange. It is also probable that a combination of these factors is re-sponsible for the observed change in morphology. The ability of P.antarctica to form colonies following release from the ice in springmay thus contribute to its dominance within phytoplankton commu-nities during spring in many areas. One of the characteristic morphol-ogies of Phaeocystis is the ‘five-rayed star arrays’ (Baumann et al.,1994). In the ice tank these were only produced during ice-free con-ditions during the light treatment but the reason for this is notknown. Interestingly, cells that were completely enveloped in thethreads had lost both flagella (Fig. 6C) and cells that were notenveloped still possessed flagellum (Fig. 6B,D). This may suggestthat periods of melt (i.e. spring) could induce vesicle rupture thereby

14 F. Kennedy et al. / Journal of Experimental Marine Biology and Ecology 429 (2012) 7–14

releasing threads and effectively establishing colonies throughentanglement.

Acknowledgements

Wewould like to acknowledge financial assistance from an AustralianAntarctic Science grant and laboratory assistance from Helen Bond. [SS]

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