ALBERT

Description: The fluorophore [2‐(4‐pyridyl)‐5{[4‐dimethylaminoethyl‐aminocarbamoyl‐methoxy]phenyl}oxazole], in short PDMPO, is incorporated in newly polymerized silica in diatom frustules and thereby provides a tool to estimate Si uptake, study diatom cell cycles but also determine mortality‐independent abundance‐based species specific‐growth rates in cultures and natural assemblages. In this study, the theoretical framework and applicability of the PDMPO staining technique to estimate diatom species specific‐growth rates were investigated. Three common polar diatom species, Pseudo‐nitzschia subcurvata, Chaetoceros simplex, and Thalassiosira sp., chosen in order to cover a broad range of species specific frustule and life‐cycle characteristics, were incubated over 24 h in control (no PDMPO) and with 0.125 and 0.6 μM PDMPO addition, respectively. Results indicate that specific‐growth rates of the species tested were not affected in both treatments with PDMPO addition. The specific‐growth rate estimates based on the PDMPO staining patterns (μPDMPO) were comparable and more robust than growth rates estimated from the changes in cell concentrations (μcc). This technique also allowed to investigate and highlight the importance of the illumination cycle (light and dark phases) on cell division in diatoms.

Description: The fluorophore [2-(4-pyridyl)-5{[4-dimethylaminoethyl-aminocarbamoyl)-methoxy]phenyl}oxazole], in short PDMPO, is incorporated in newly polymerized silica in diatom frustules and thereby provides a tool to estimate Si uptake, to study diatom cell cycles but also to determine mortality-independent abundance-based species specific growth rates in cultures and natural assemblages. In this study, the theoretical framework and applicability of the PDMPO staining technique to estimate diatom species specific growth rates were investigated. Three key polar diatom species, Pseudonitzschia subcurvata, Chaetoceros simplex and Thalassiosira sp., chosen to cover a broad range of species-related frustule and life-cycle characteristics, were incubated over 24 hours in control (no PDMPO) and with 0.125 µM and 0.6 µM PDMPO addition, respectively. The main assumptions tested during this study were: 1) Addition of PDMPO does not affect division rates. 2) Newly divided cells (daughter cells) can be readily recognized by their fluorescent valves and PDMPO is taken up only in newly formed valves. 3) The populations do not divide synchronously (here the impact of light-dark cycles on division was also included). Assumptions 1 and 2 were tested by comparing cell concentration-based growth rates with those based on PDMPO stain in control incubations and in incubations where PDMPO was added. This was carried out for P. subcurvata (Ps), C. simplex (Cs) and Thalassiosira sp. (Ts) acclimated to 20 µmol photon/m²/s at 0.125 µM (all species) and 0.6 µM (for Ps and Cs) PDMPO final concentration. The impact of PDMPO addition was further tested on Thalassiosira sp. acclimated at 110 µmol photon/m²/s at both 0.125 µM and 0.6 µM PDMPO final concentration.

Description: To ensure balanced growth and stable conditions, all cultures were acclimated in semi-continuous batch cultures for at least nine generations before starting the experiments. Following acclimation, the experimental incubations were carried out in two consecutive phases: 1) To ensure that cells were acclimated and growing exponentially, cultures were transferred into new media at the end of the exponential growth phase for another nine generations. 2) Following acclimation, cultures were diluted into 8 or 12 incubations bottles (replicate incubations) containing new media. In the first days after transfer, cell concentrations were monitored to ensure exponential growth ("Control phase"). After this short period, [2-(4-pyridyl)-5{[4-dimethylaminoethyl-aminocarbamoyl)-methoxy]phenyl}oxazole] (PDMPO, LysoSensor Yellow/Blue DND-160, Thermo Fisher Scientific, Waltham, MA, USA) was added to four or eight (depending on species and experiment) replicate bottles while the remaining four bottles were incubated without stain addition (controls). Experiments were terminated and sampled 24 h (full light-dark cycle) after PDMPO addition. Daily samples for cell enumeration, fixed with acidic Lugol's solution (around 1% f.c.), were taken during the control phase. After addition of PDMPO, samples for cell enumeration and PDMPO analysis were taken at the time of stain addition (t0) and 24 hours later (t24). Further samples were taken at the beginning (if different from t0) and end (if different from t24) of both light and dark cycles, respectively. Samples were fixed with 2% (f.c.) hexamine-buffered formalin and stored in glass vials in the dark at 4°C until analysis. To determine cell abundance, 10 mL of undiluted or diluted fixed sample (from 3 to 4 independent replicate bottles each) were settled in a 10 ml Utermöhl sedimentation chamber (HYDRO-BIOS, Kiel, Germany). At least 300 cells were counted with a Zeiss Axiovert 40C inverted light microscope or a Zeiss Axiovert 200 epifluorescence microscope. Samples with PDMPO were counted with the aforementioned epifluorescence microscope equipped with a long pass filter (Zeiss Filter set 02; ex: G365, bs: FT395; em: LP420). Samples were counted at 200x to 630x magnification depending on species and intensity of the PDMPO signal.

Description: While in culture, specific growth rate (μ) can be easily determined through cell counts (N0 and Nt). This is not the case for incubations with natural assemblages, where grazers and other processes lead to cell loss and consequently an underestimation of Nt. Incubations with [2-(4-pyridyl)-5{[4-dimethylaminoethyl-aminocarbamoyl)-methoxy]phenyl}oxazole] (PDMPO) overcomes this issue by allowing the determination of N0 (Nt=N0×e^(μt) (Eq. 1)) based on the relative proportion of PDMPO stained cells (newly divided) and unstained cell at the end of an incubation as follows: Given Nt, the total cell number of a species from a subsample at a time t after PDMPO addition. Nt=nt+nt', with nt the total number of non-(PDMPO) stained cells of the species in the same subsample (cells that did not divide yet), and nt' the total number of cells with one PDMPO stained valve (cells issued from the first division after PDMPO addition to the culture media). All things being equal, the population (N0) at the time when PDMPO was added that gave rise to Nt should be N0=nt+((nt')/2)=Nt-((nt')/2) (Eq. 2). Growth rates from cells counts were estimated using (Eq. 1), while growth rates using PDMPO were calculated from the number of non-stained (nt), half-stained (nt') and fully stained (nt'') cells using μ[d-1]=ln((nt+nt')/(nt+(nt')/2))×(1/t) (Eq. 3) or μ[d-1]=ln((nt'+nt'')/((nt')/2))×(1/t) (Eq. 4). Cells attached to each other (potentially in the final phase of division) were also considered as non-stained, half-stained and fully-stained individuals, respectively, based on the presence/absence of a PDMPO signal on the valves.

Description: The fluorophore [2-(4-pyridyl)-5{[4-dimethylaminoethyl-aminocarbamoyl-methoxy]phenyl}oxazole], in short PDMPO, is incorporated in newly polymerized silica in diatom frustules and thereby provides a tool to estimate Si uptake, study diatom cell cycles but also determine mortality-independent abundance-based species specific-growth rates in cultures and natural assemblages. In this study, the theoretical framework and applicability of the PDMPO staining technique to estimate diatom species specific-growth rates were investigated. Three common polar diatom species, Pseudo-nitzschia subcurvata, Chaetoceros simplex, and Thalassiosira sp., chosen in order to cover a broad range of species specific frustule and life-cycle characteristics, were incubated over 24 h in control (no PDMPO) and with 0.125 and 0.6 μM PDMPO addition, respectively. Results indicate that specific-growth rates of the species tested were not affected in both treatments with PDMPO addition. The specific-growth rate estimates based on the PDMPO staining patterns (μPDMPO) were comparable and more robust than growth rates estimated from the changes in cell concentrations (μcc). This technique also allowed to investigate and highlight the importance of the illumination cycle (light and dark phases) on cell division in diatoms.