Diatom Research, 2015Vol. 30, No. 1, 87–92, http://dx.doi.org/10.1080/0269249X.2014.960006

Silicon limitation induces colony formation in the benthic diatom Nitzschia cf. pusilla(Bacillariales, Bacillariophyceae)

M. SOLEDAD FUENTES1,2, SHANNON L. MESECK1∗, GARY H. WIKFORS1 & DIBA KHAN-BUREAU3

1Department of Commerce, National Oceanographic and Atmospheric Administration, Connecticut, USA2Department of Physiology, Fort Myers, Algenol Biofuels, Florida, USA3Department of Natural Resources and the Environment and Department of Ecology and Evolutionary Biology, University ofConnecticut, Connecticut, USA

While investigating the effects of silicon limitation on the diatom Nitzschia cf. pusilla, we observed, along with expected lower maximumcell density, colony formation. Cells attached in stacks that increased in number, reaching an average of 30 ± 1.8 (mean ± SE) cells perstack. Cells grown under silicon-replete conditions formed short stacks (three to four cells) in the stationary phase. When silicon-depletedcultures were split and silicate was added to half of the split cultures, cell stacks decreased in the cultures to which silicate was added.These results suggest that N. pusilla may possess an adaptive strategy to survive and maintain growth under silicon limitation.

Keywords: frustule, meroplanktonic, microphytobenthos, nutrient limitation

IntroductionDiatoms constitute the predominant group of phytoplank-ton in marine environments, contributing approximately40% of total marine primary production (Nelson et al.1995). Given their characteristic siliceous frustules, sili-con (Si) is an essential macronutrient for the growth andreproduction of diatoms. Because they are one of the mostsuccessful microalgal groups, this silicified cell wall mustbe a contributing factor to their success, providing fitnessadvantages. Some hypothesized functions of the frustulein diatoms include defence, light dispersion and sinking-rate regulation (Davidson et al. 1994, Ham et al. 2003,Raven & Waite 2004). Furthermore, cell-wall silicificationhas a low energetic cost compared with lignification or sac-charide polymerization, processes that are 10- to 20-foldmore energetically expensive (Raven 1983). Even thoughSi is one of the most common elements in the earth’s crust(Reynolds 2006), after spring and summer diatom blooms,dissolved Si in marine systems often declines to concen-trations below 2 μM, which for most diatom species islimiting for growth (Riegman et al. 1990, Ragueneau et al.1994, Finkel & Kotrc 2010).

One of the first responses of diatoms to Si deficiency isa decline in growth-rate. When Si is deficient, cell divisionis arrested by preventing the production of new daughter-cell valves (Martin-Jézéquel et al. 2000), via several Si-dependent arrest points during the cell cycle: early in G1

*Corresponding author. Email: shannon.meseck@noaa.gov

(Received 24 December 2013; accepted 25 July 2014)

(e.g., formation of the siliceous setae in Chaetoceros sp.(Ehrenberg, 1844), at the G1/S boundary (Si dependencyfor DNA synthesis), and at G2/M (when new valves areforming) (Brzezinski et al. 1990, Martin-Jézéquel et al.2000).

However, diatoms have developed adaptive responsesto the environmental instability in Si availability, suchas phenotypic variations that allow them to mitigate thestress of nutrient depletion, thereby surviving adverse Siconditions (Smayda & Boleyn 1965, Takabayashi et al.2006). Diatom polymorphism is species-specific, a func-tion of genetic diversity that is regulated by environmentalconditions. Phenotypic variation resulting from Si defi-ciency includes thinner frustules (Thalassiosira nordenski-oldii Cleve [Paasche 1975]), reduced numbers of siliceousspines (Chaetoceros debilis Cleve [Harrison et al. 1977])and colony-size regulation (Werner & Stangier 1976, Kling1993). Because species identification of diatoms is basedon valve morphology, phenotypic plasticity can lead to tax-onomic confusion and the existence of different morphsmust therefore be considered when attempting species-level identifications. Nonetheless, when recognized, poly-morphism in diatom species can be used as a bio-indicatorof environmental status (Tapia 2008).

In the present study, we describe the effects of Si defi-ciency on the morphology of the marine diatom Nitzschiacf. pusilla (Grunow) Hustedt in batch culture.

© 2014 The International Society for Diatom Research

88 Fuentes et al.

Materials and methodsBacteria-free cultures of N. cf. pusilla, strain O-7 ( =CCMP560), isolated by Guillard in 1956 from a brackishtidal pond (Oyster Pond, Martha’s Vineyard, MA, USA),were obtained from the Milford Laboratory MarineMicroalgal Culture Collection. Cultures were maintainedin 1 L polystyrene culture flasks at 16 ± 1°C on a 12 h:12h light/dark cycle and under a light intensity of 110 μmolphotons m−2 s−1. Si-replete (control) and Si-depleted batchcultures (N = 4 per treatment) were inoculated with astarting cell density of c. 20 000 cells mL−1. A phosphate-depleted treatment (with 6.18 ± 1.22 μM [ ± SD] phos-phate compared with 40.76 ± 1.08 μM phosphate in thecontrol) was also added to discount stack formation underother conditions. Nitzschia cf. pusilla cells were grown inSargasso seawater enriched with nutrients at f/2 (Guillard1975) concentrations and under the same conditions as theinoculum culture. The Si concentration in the control treat-ment was 107.8 ± 1.4 μM ( ± SD) and in the Si-depletedtreatment was 33.7 ± 0.1 μM. Initial and final nutrientconcentrations were quantified using a TRAACS 800 auto-analyzer (Bran and Luebbe, Delavan, WI, USA), followingthe protocol described by Hansen & Koroleff (1999). Dis-solved phosphate concentrations were determined usingthe phosphomolybdenum complex (detection limit 0.08μmol L−1). Silicon determination was based upon the for-mation of a blue silicomolybdic complex (detection limit0.10 μM). Nitrate + nitrite were determined only to ensurethat the media were not nitrogen-depleted during the exper-iment. Nitrate + nitrite concentrations never fell below300 μM for any of the treatments. Growth of the cultureswas measured by flow cytometry (FACScan, BD Bio-Sciences, San Jose, CA, USA) every day for nine days.Using a blue laser and a flow rate of 60 μl min−1, each sam-ple was counted for 30 s. Cells were distinguished basedupon particle size (forward scatter) and chlorophyll fluo-rescence (FL3 detector, > 650 nm). The viability of thecells was assessed on day 8, also by flow cytometry, usingthe nucleic acid stain SYTOX® Green (Invitrogen, Molec-ular Probes), which is detected by the FL1 flow cytometerdetector (530 nm wavelength). SYTOX Green stains deadcells as it only permeates compromised cell membranes.Cell size in a fresh sample preparation was measured at day8 by counting and photographing 100 cells using a ZeissAxioscop 2 MOT Plus microscope (Carl Zeiss MicroscopyLLC, Thornwood, NY, USA) at × 40 magnification.

Scanning electron microscopy (SEM) preparationTo enhance attachment of the fragile diatoms to a smoothsubstratum, a drop of sample was applied to the sur-face of a silica wafer that had been freshly coated with0.1% poly-L-lysine. After 15 min, wafers were trans-ferred to a fixative containing 1.5% glutaraldehyde and1.5% paraformaldehyde in 0.1 M sodium cacodylate with0.375 M sodium chloride (NaCl), pH 7.7, for 1 h at

room temperature. Wafers were then washed gently threetimes in 0.1 M sodium cacodylate with 0.375 M NaCl, 20min each, and placed in a buffered 2% osmium tetrox-ide solution overnight at 4°C. The next day, wafers werewashed three times in distilled water, dehydrated throughan ascending ethanol series, and finally dried by criticalpoint drying (CPD). Stubs were coated for 1 min at 1.8kV with gold/palladium using a Polaron sputter coater.The stubs were viewed with the field emission Leo/ZeissDSM 982 (Carl Zeiss Microscopy LLC) SEM located atthe University of Connecticut Electron Microscopy lab.

ResultsAfter two days, Si-depleted cultures had 33% lower cellcounts than control cultures, showing signs of starvation(Fig. 1), but cells continued growing until day 6, whenthey reached the stationary phase. The maximum cell den-sity of the Si-depleted cultures (175 000 ± 17 700 cellsmL−1 [ ± SE]) was reached on day 7, at which time themean cell density of the control cultures was almost twiceas high (364 000 ± 5700 cells mL−1 [mean ± SE]). Con-trol cultures reached stationary phase after eight days ofcontinuous growth. At this point, the mean cell count incontrol cultures was more than double that of Si-depletedcultures (Fig. 1).

At the time of inoculation, only solitary cells wereobserved (Fig. 2a). In the Si-depleted cultures, however,cells formed stacks after only one day of cultivation (Fig.2b and Fig. 3), significantly different from the control cul-ture (P < 0.01). Numbers of cells per stack increased pro-gressively until the cultures reached the stationary phase(day 6), when no solitary cells were observed. Also byday 6, Si-depleted cells were shorter than cells in the con-trol treatment (20.4 ± 0.5 and 22.0 ± 0.3 μm [ ± SE],respectively; P < 0.01 for 100 cells measured in eachtreatment). The average number of cells per stack in sta-tionary phase (day 7) was 30.00 ± 1.81 ( ± SE) (Fig. 3). Itis possible that Si-depleted cultures had even larger stacksthat were not accounted for due to breakage during sample

Fig. 1. Nitzschia cf. pusilla growth curve under Si-depleted (�)and control (�) conditions.

Si and colony formation in Nitzschia pusilla 89

Fig. 2. Cells of N. cf. pusilla grown under Si-replete conditions(control) (A) and Si-depleted conditions (B).

preparation. No cell stacks were observed in the controltreatments until the cultures reached the stationary phaseon day 8, when cells started forming stacks of three tofour cells. However, stacks in the control were significantlyshorter than in Si-depleted cultures (P < 0.01). Stacks ofcells were not observed under phosphate deficiency (datanot shown).

SYTOX Green fluorescence showed no differencebetween stained and unstained samples (i.e., no dead cellswere detected) and there was also no difference in cell via-bility between Si-depleted and control cultures (F = 3.35;P = 0.08).

Even though SEM images of Si-depleted culturesshowed dramatic cell-stacking, no extracellular polysac-charides were detected by iodine staining, either in cells instacks or solitary cells. However, incomplete frustule sepa-ration seemed to be the cause of cell stacking (Fig. 4). SEMimages also revealed valve malformation, possibly due topoor silicification.

Nine days after inoculation, the Si-depleted cultureshad decreased from 33.70 ± 0.10 μM ( ± SD) Si to nodetectable Si, whereas the control had decreased from107.80 ± 1.40 to 49.26 ± 0.93 μM ( ± SD) in the control.Silicate (c. 100 μM) was then added to the Si-depleted cul-tures. After another day, the Si re-enriched cultures divided(333 000 ± 10 400 cells mL–1 [ ± SE]), the mean lengthof these cells was no longer significantly different fromthe length of solitary cells in the control treatment (22.2± 0.37 and 22.0 ± 0.33 μm [ ± SE], respectively), and

Fig. 3. Number of cells per stack over time in Si-depleted (�)and control (�) cultures.

no stacking was observed. In summary, when silicate wasadded and cell division resumed, cells recovered in lengthas they divided from within the stacks.

DiscussionThe results of this study show that cells of N. cf. pusilla(O-7) form colonies as a response to Si deficiency. Fur-thermore, the fact that Si-deficient cells were shorter sug-gests silicification was limited to the areas immediatelyaround the protoplast. Lighter silicification, would prob-ably reduce the number of girdle bands produced and pre-vent cells from separating. We interpret these observationsas evidence that cells of this species growing under Si defi-ciency continue to divide, although at lower rate, but donot separate from each other, perhaps to minimize the Sicell quota.

Microalgae exhibit considerable morphological plas-ticity in response to environmental factors (Trainor et al.1971, 1976, Morales & Trainor 1997) but, currently,very little is known about the ecological significance ofcolony formation or its causes. Previous studies havedescribed that kairomones from the grazer Daphnia inducecolony formation in the green algae Desmodesmus andScenedesmus (Hessen & Van Donk 1993). The diatomSkeletonema marinoi Sarno et Zingone avoids copepodgrazing by modifying chain formation in response to cope-pod chemical cues (Bergkvist et al. 2012). In PhaeocystisLagerheim, however, colony formation prevents grazing(Smetacek et al. 2004). These observations suggest, there-fore, that colony length regulation might be an adaptiveresponse to grazing to reduce mortality in those microal-gae. In the prymnesiophyte Phaeocystis pouchetii Hariot,however, studies have linked variation in colony formationand colony length regulation to nutrient concentrations.Veldhuis & Admiraal (1987) reported colony formation ofP. pouchetti when dissolved phosphate was below 1 μM,whereas, above this concentration colony formation wasinhibited. Tilman & Kilham (1976) reported a change inthe number of cells per colony of the diatom Asterionellaformosa Hassall under Si deficiency. Cells-per-colonyincreased (up to 20 cells per colony) with decreasing Siconcentration. In the same species, Kling (1993) reportedcell-size reduction associated with Si limitation in the pres-ence of excess P. Werner & Stangier (1976) demonstratedthe participation of Si in the regulation of colony formationin the centric marine diatom Bellerochea maleus f. bian-gulata Peragallo; both low temperature and low Si con-centration increased mean colony size. Teng (1984) alsosuggested an interaction between Asterionella glacialisCastracane colony formation and organic substances, tracemetals, and Si. Takabayashi et al. (2006) showed thatSkeletonema costatum (Greville) Cleve increases chainlength with higher temperatures and nutrient availability.All this evidence suggests that, under different scenariosand environmental conditions, colony formation might

90 Fuentes et al.

Fig. 4. SEM of N. cf. pusilla. (A, B) Si-depleted cultures forming colonies (scale bar 10 and 20 μm, respectively). (C, D) Incompletefrustule separation (scale bar 5 μm) and in Si-depleted cultures. (E, F) Detail of Si-depleted N. pusilla valves (scale bar 2 μm).

have different ecological significance but the same funda-mental purpose for different species, which is enhancingfitness. In this study, a typically unialgal diatom speciesappeared to deal with Si stress by altering morphology,perhaps to minimize Si requirements. Even though theimportance of Si for the regulation of colony formation andsize has been indicated in previous studies, as a strategy tomaintain growth under Si deficiency colony formation indiatoms has not previously been described. This strategymight confer an adaptive advantage allowing N. cf. pusillato survive under Si-depleted conditions in areas similarto its source locality (in southern New England, USA inbrackish tidal ponds). With this strategy, populations of N.cf. pusilla would have more cells ready to proliferate andoutcompete other microalgal populations when conditionsimproved.

There are several hypotheses that might explain howcolony formation under Si deficiency could increase

fitness. Colony formation under Si deficiency might lead toan increase in sinking rate (a consequence of Stokes’s law),returning populations to the sediment surface where regen-erated Si might be available. However, because N. pusillais benthic, this explanation might not apply. Benthic algaecan access nutrients from the upper water layer, but suchaccess is often limited by still water (boundary layer) (Mul-holland 1996). Could chain formation enable the diatomsto break through the benthic boundary layer for betteraccess to nutrients? Although further testing is needed toconfirm this hypothesis, such adaptive behaviour, drivenby environmental conditions, could allow this species toovercome nutrient discontinuities in the environment.

AcknowledgementsWe thank Jennifer L. Alix, Kimberly Dickinson, Kathy ShuoZhai, and Scott M. Hawley for their assistance during the experi-ment. We also thank Dr. Marie Cantino and Steve Daniels in the

Si and colony formation in Nitzschia pusilla 91

UConn EM lab for their support and Dr. Eduardo Morales for hishelpful suggestions. This project was supported by the NOAANational Aquaculture Program, through a National ResearchCouncil Post-Doctoral Fellowship to M.S.F., and by the North-east Fisheries Science Center of the National Marine FisheriesService.

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