Waiting
Login processing...

Trial ends in Request Full Access Tell Your Colleague About Jove

Biology

Measuring Photophysiology of Attached Stage of Colacium sp. by a Cuvette-Type Fast Repetition Rate Fluorometer

doi: 10.3791/63108 Published: November 12, 2021
Takehiro Kazama1,2,3, Kazuhide Hayakawa4, Koichi Shimotori1,2, Akio Imai1

Abstract

Fast repetition rate fluorometer (FRRf) is a beneficial method for measuring photosystem II (PSII) photophysiology and primary productivity. Although FRRf can measure PSII absorption cross-section (σPSII), maximum photochemical efficiency (Fv/Fm), effective photochemical efficiency (Fq′/Fm), and non-photochemical quenching (NPQNSV) for various eukaryotic algae and cyanobacteria, almost all FRRf studies to date have focused on phytoplankton. Here, the protocol describes how to measure PSII photophysiology of an epizoic alga Colacium sp. Ehrenberg 1834 (Euglenophyta), in its attached stage (attached to zooplankton), using cuvette-type FRRf. First, we estimated the effects of substrate zooplankton (Scapholeberis mucronata O.F. Müller 1776, Cladocera, Daphniidae) on baseline fluorescence and σPSII, Fv/Fm, Fq′/Fm, and NPQNSV of planktonic Colacium sp. To validate this methodology, we recorded photophysiology measurements of attached Colacium sp. on S. mucronata and compared these results with its planktonic stage. Representative results showed how the protocol could determine the effects of calcium (Ca) and manganese (Mn) on Colacium sp. photophysiology and identify the various effects of Mn enrichment between attached and planktonic stages. Finally, we discuss the adaptability of this protocol to other periphytic algae.

Introduction

Chlorophyll variable fluorescence is a useful tool for measuring algal photosystem II (PSII) photophysiology. Algae respond to various environmental stresses, such as excess light and nutrient deficiency, by altering their PSII photophysiology. Fast repetition rate fluorometer (FRRf) is a common method for measuring PSII photophysiology1,2 and estimating primary productivity1,3,4, which enables monitoring phytoplankton PSII photophysiology, as well as primary productivity across wide spatial and temporal scales5,6,7. FRRf can simultaneously measure PSII (σPSII) absorption cross section, reaction center ([RCII]) concentration, maximum photochemical efficiency (Fv/Fm), effective photochemical efficiency (Fq′/Fm), and non-photochemical quenching (NPQNSV) (Table 1). In general, Fv/Fm and Fq′/Fm are defined as PSII activity8, while NPQNSV is defined as relative heat-dissipated energy9.

Importantly, single turnover (ST) flashes of FRRf fully reduce the primary quinone electron acceptor, QA, but not the plastoquinone pool. Conversely, multiple turnover (MT) flashes from a pulse amplitude modulation (PAM) fluorometer can reduce both. The ST method has a clear advantage over the MT method when identifying the possible origins of NPQNSV by simultaneously measuring recovery kinetics of Fv/Fm, Fq′/Fm, NPQNSV, and σPSII10. To date, several types of FRRf instruments, such as submersible-type, cuvette-type, and flow-through-type, are commercially available. The submersible-type FRRf enables in situ measurements in oceans and lakes, while the cuvette-type FRRf is suitable for measuring small sample volumes. The flow-through type is commonly used to continuously measure the photophysiology of phytoplankton in surface waters.

Given the development of PAM fluorometers, including the cuvette-type, for a broad range of subjects11, PAM fluorometers are still more common than FRRfs in algal photophysiology research12. For example, although the sample chamber structure and cuvette capacity between these tools only differs slightly, the cuvette-type PAM has been applied to phytoplanktons13,14,15, benthic microalgae16,17,18, ice algae19, and epizoic algae20, while the cuvette-type FRRf has been applied primarily to phytoplanktons21,22,23 and a limited number of ice algal communities24,25. Given its effectiveness, cuvette-type FRRf is equally applicable to benthic and epizoic algae. Therefore, expanding its application will provide considerable insight into PSII photophysiology, particularly for lesser-known epizoic algal photophysiology.

Epizoic algae have received little attention, with few studies examining their PSII photophysiology20,26, most likely due to their minor roles in aquatic food webs27,28. However, epibionts, including epizoic algae, can positively influence zooplankton community dynamics, such as increasing reproduction and survival rates29,30, as well as negatively impact processes, such as increasing sinking rate29,31 and vulnerability to visual predators32,33,34,35,36. Therefore, exploring the environmental and biological factors controlling epibiont dynamics in zooplankton communities is crucial.

Among epizoic algae, Colacium Ehrenberg 1834 (Euglenophyta) is a common, freshwater, algal group32,37,38,39 with various life stages, including attached (Figure 1A-D), non-motile planktonic (Figure 1E,F), and motile planktonic stages40,41. During the non-motile planktonic stage, cells live as single-cell planktons, aggregated colonies, or one-layer sheet colonies, covered by mucilage42. In the attached stage, Colacium sp. uses mucilage excreted from the anterior end of the cell37,39,41 to attach to substrate organisms (basibionts), particularly microcrustaceans41,43. Their life cycle also involves detaching from the molted exoskeleton or dead basibiont and swimming with their flagella to find another substrate organism39. Both planktonic and attached stages can increase their population size by mitosis40. Although their attached stage is hypothesized to be an evolutionary trait for gathering resources, such as light44 and trace elements41,45,46, or as a dispersion strategy27, little experimental evidence is available about these aspects37,41,44 and the key attachment mechanisms are largely unknown. For example, Rosowski and Kugrens expected that Colacium obtains manganese (Mn) from substrate copepods41, concentrated in the exoskeleton47.

Here, we describe how to measure PSII photophysiology of planktonic algae and the related application method for targeting attached algae (attaching to zooplankton) with Colacium sp. cells using the cuvette-type FRRf. We use the Act2 system equipped with three light-emitting diodes (LEDs) that provide flash excitation energy centered at 444 nm, 512 nm, and 633 nm48. Here, 444 nm (blue) corresponds to the absorption peak of chrophyll a (Chl-a), while 512 nm (green) and 633 nm (orange) correspond to the absorption peaks of phycoerythrin and phycocyanin, respectively. The fluorescent signal detection peak is 682 nm with 30 nm half bandwidth. Since it is difficult to find the planktonic stage of Colacium sp. in natural environments, their attached stage was collected for the experiments. Among the numerous substrate organisms,Scapholeberis mucronata O.F. Müller 1776 (Branchiopoda, Daphniidae; Figure 1A,B,G) is one of the simplest to handle due to their slow swimming speed, large body size (400-650 µm), and unique behavior (hanging upside down on the water surface). Therefore, this protocol uses Colacium sp. attached on S. mucronata as a case study of the Colacium-basibiont system. To avoid fluorescence derived from the gut contents, S. mucronata was starved. As a previous study reported that the fluorescence signal from gut contents (ingested algae) displays a five-fold decrease after 40 min49, we expected that 90 min starvation would be enough to minimize the possibility of gut content fluorescence affecting the FRRf measurement with minimum effects of experimental stress to Colacium sp., such as nutrient deficiency. Furthermore, this protocol was applied to clarify the attaching mechanism of Colacium sp. and determine how two metals, calcium (Ca) and manganese (Mn) affect the photophysiology of both planktonic and attached stages. Calcium plays key roles in the photosynthetic pathways50 in multiple ways, and both metals are required to construct the oxygen-evolving complexes of the PSII51. As calcium and manganese are highly concentrated in the carapace of crustacean zooplankton47, we hypothesize that Colacium sp. photophysiology might respond more prominently to Ca and Mn enrichment during the planktonic stage if this life stage obtains these elements from S. mucronata during the attached stage.

Subscription Required. Please recommend JoVE to your librarian.

Protocol

1. Sampling

  1. Collect lake water from the surface by a bucket. To target Colacium sp. attached to S. mucronata (Figure 1A-C) filter 0.5-10 L of lake water using a 100 µm nylon mesh net52.
    NOTE: S. mucronata often densely aggregate in shallow, eutrophic, muddy water, such as among reed (phragmites) areas.
  2. Store the concentrated samples in 500 mL plastic bottles with 350 mL of lake water. Keep in dark conditions.
  3. In the laboratory, pour the sample water into a 500 mL beaker and allow it to settle for a few minutes.
  4. Filter the lake water through a 0.2 µm pore-size filter.
  5. Pick up S. mucronata individuals using a pipette under an optical microscope at 100x magnification. Perform species identification according to Błędzki and Rybak53.
  6. Transfer them into a drop of 0.2-µm filtered lake water (FLW) placed on a glass slide.
    NOTE: S. mucronata may swim to the surface or attach to the beaker wall.
  7. Check S. mucronata under light microscopy.
  8. Wash S. mucronata individuals using FLW (3 drops or more) to prevent contamination from other organisms (Figure 2).
  9. Keep S. mucronata at an in situ temperature in a growth chamber under 40 µmol photon·m−2·s−1.

2. Effects of S . mucronata on baseline fluorescence

  1. S. mucronata cultivation
    1. Pick up S. mucronata individuals using a pipette under an optical microscope at 100x magnification and wash using FLW, as in step 1.8.
    2. Aerate tap water using an electric air pump via an air stone for at least 1 week. Pour 300 mL of aerated tap water into a 350 mL glass jar.
    3. Feed Chlorella (1 mg C·L−1) and maintain at 20 °C under 40 µmol photon·m−2·s−1 in a growth chamber.
    4. After approximately 14 days, pick up 5-30 individuals using a pipette under an optical microscope at 100x magnification and inoculate them into 300 mL of clean, aerated tap water to keep the medium fresh.
  2. Setting up FRRf
    1. Launch the Act2Run software.
    2. Click on the Options tab and select Act2 FLC (white LEDs) in Mode of Operation to set the actinic LEDs color.
    3. Click on the value of Dark at step 1 of the settings of the fluorescent-light curve in the main window, and type 30 to set the duration of the dark period (Figure 3A).
    4. Click on the LED combination B, C, and D to turn off the green and orange LEDs (Figure 3C).
    5. Click on the value of Fets and Pitch under Sat, and type 100 and 2, respectively, to set the number and pitch of flashlet in the saturation phase (Figure 3D).
    6. Click on the value of Fets and Pitch under Rel, and type 40 and 60, respectively, to set the number and pitch of flashlet in the relaxation phase (Figure 3E).
    7. Activate the water jacket pump by clicking on During FLC (Figure 3F) to control sample temperature during the measurement.
    8. Activate by clicking Auto-LED and Auto-PMT (Figure 3F).
    9. Click on Synchronize to connect FRRf-Act2.
  3. FRRf measurements
    1. To examine the effects of zooplankton individuals on baseline fluorescence, prepare adult S. mucronata (body size 400-650 µm) from the culture in steps 2.1.1-2.1.4 without any attached organisms.
    2. To avoid fluorescence from the gut contents, starve the individuals in FLW at 20°C for at least 90 mins.
    3. Pour 1.5 mL of FLW into a cuvette. Pick up 0, 1, 5, and 10 S. mucronata individuals using a pipette under an optical microscope at 100x magnification.
    4. Transfer S. mucronata individuals into the cuvette and add FLW to bring the sample up to 2 mL.
    5. Acclimate under low light (1-10 µmol photon·m−2·s−1) at 20 °C for 15 min before FRRf measurement.
    6. Click on Act2 Run to start the measurement. Repeat the measurements >3 times per sample.
    7. Read the Fo value from the result plot (Figure 4).

3. Effects of substrate organism on Chl- a fluorescence

  1. Colacium sp. cultivation
    1. Prepare the FLW and AF-6 medium54 for cultivation (Table 2).
    2. Collect Colacium sp. attached to S. mucronata as in steps 1.1 and 1.2 and, keep at in situ temperature in a growth chamber.
    3. Pick up Colacium sp. with a molted carapace (Figure 1D) using a pipette under an optical microscope at 100x magnification. Wash them with FLW, as in step 1.8.
    4. Aseptically inoculate Colacium sp. and AF-6 medium in a 10 mL glass tube on a clean bench.
    5. Maintain the culture at in situ temperature under 200 µmol photon·m−2·s−1 in a growth chamber. Shake the glass tube gently by hand at least once per day to prevent cell settlement.
      NOTE: To keep the attenuation effect of aggregated colonies as low as possible, check the colonies under a microscope prior to FRRf measurement. Cell aggregation may cause dense colonies and affect algal photophysiology55.
  2. FRRf measurements
    1. To examine the effects of zooplankton individuals on Chl-a fluorescence from Colacium sp., prepare adult S. mucronata (body size 400-650 µm) without any attached organisms.
    2. To avoid fluorescence from the gut contents, starve the individuals in FLW for at least 90 min.
    3. Set up a cuvette-type fast repetition rate fluorometer (FRRf).
    4. Pour a 1.5 mL subsample of precultured Colacium sp. into a cuvette. Transfer 0, 5, 10, and 15 S. mucronata individuals into these cuvettes and add 2 µm of filtered medium to bring the sample up to 2 mL.
    5. Acclimate under low light (1-10 µmol photon·m−2·s−1) at 20 °C for 15 min before taking the FRRf measurement.
      NOTE: Maintain the samples at incubation temperature during measurements
    6. Click on Act2 Run to start the measurement. Repeat the measurements >3 times per sample.
    7. Read the Fand Fm values from the result plot (Figure 4).
      NOTE: Check the PSII value (Table 1), which shows whether the LED power is within the optimal range to estimate the PSII parameters correctly. When the Auto-LED is activated, Act2run system controls the LED power to achieve an optimal PSII range (0.042-0.064). The experimental PSII cut-off value was defined at 0.03 and 0.08 in a previous study48.
    8. To correct the baseline fluorescence22, filter the culture medium using a 0.2-µm pore-size filter and measure the fluorescence. Subtract FO of the baseline sample from FO and Fm of Colacium sp., or modify the Blank correction value in the Settings in the Options tab.

4. Photophysiology of Colacium sp. (attached stage)

  1. Isolate S. mucronata individuals with Colacium sp. using a pipette under an optical microscope.
  2. Wash S. mucronata using FLW, as in step 1.8.
  3. Transfer S. mucronata into a 100 mL of FLW. For starvation, keep under dark conditions at in situ temperature for 90 min.
  4. Pour 1.5 mL of FLW into a cuvette.
  5. Transfer ~10 S. mucronata individuals with Colacium sp. into a cuvette. For measurements, more than 100 Colacium cells per 2 mL are needed. Add FLW to bring the sample up to 2 mL.
  6. Acclimate under low light (1-10 µmol photon·m−2·s−1) at in situ temperature for 15 min. Measure Chl-a fluorescence as in steps 3.2.6-3.2.8.
  7. To enumerate the number of attached cells, fix the sample with glutaraldehyde (2% final volume) after taking the FRRf measurement. Take pictures at several focal depths and positions of S. mucronata under a light microscope.

5. Photophysiology of Colacium sp. (planktonic stage)

  1. Cultivate sampled Colacium sp. in AF-6 medium at in situ temperature as in steps 3.1.1-3.1.5.
  2. For the stationary phase, take 2 mL of cultured Colacium sp. and pour into a cuvette.
  3. Acclimate under low light (1-10 µmol photon·m−2·s−1) at in situ temperature for 15 min. Measure Chl-a fluorescence as in steps 3.2.6-3.2.8.

6. Effects of Ca and Mn addition on photophysiology of Colacium sp.

  1. Effects on attached stage
    1. Isolate S. mucronata individuals with Colacium sp. using a pipette under an optical microscope. Wash using FLW, as in step 1.8.
    2. Transfer six individuals each into 12 glass beakers with 30 mL of FLW. Ensure that each beaker contains >100 Colacium sp. cells.
    3. Add 200 µmol·L−1 CaCl2·H2O (Ca treatment), 40 µmol·L−1 MnCl4 (Mn treatment), or ultrapure water (control) to each beaker. Incubate the samples under 200 µmol photon·m−2 ·s−1 at in situ temperature in a growth chamber.
    4. At 3 h and 21 h, transfer all individuals and molted skins into a cuvette with 2 mL of the medium.
    5. To examine the rapid response to increasing light, click on Up of the periods of 8 actinic light steps and type 20 (Figure 3A) to set the duration of each step in 20 s. To set the stepwise actinic light as 0, 11, 25, 44, 68, 101, 144, and 200 µmol photon·m−2·s−1, click on High E and Step Up and change the values to 200 and 34, respectively (Figure 3B).
    6. After 15 min of dark acclimation, measure Chl-a fluorescence of each sample similar to steps 3.2.6-3.2.8.
      NOTE: Verify that the PSII and PSII' values are within the optimal range (0.03-0.08)48.
  2. Effects on planktonic stage
    1. Cultivate sampled Colacium sp. in AF-6 medium at in situ temperature as in steps 3.1.1-3.1.5.
    2. Transfer the cultured Colacium sp. into FLW and acclimate at in situ temperature less than 200 µmol photon·m−2·s−1 for 3 days.
    3. Transfer 1 mL of the acclimated samples into three glass vials with 10 mL of FLW.
    4. Add 200 µmol·L−1 CaCl2·H2O (Ca treatment), 40 µmol·L−1 MnCl3 (Mn treatment), or ultrapure water (control) to vials. Incubate the samples under 200 µmol photon·m−2·s−1 at in situ temperature in a growth chamber.
    5. At 3 h and 21 h, measure Chl-a fluorescence of each sample as in steps 3.2.6-3.2.8.

Subscription Required. Please recommend JoVE to your librarian.

Representative Results

There was no significant effect of baseline fluorescence (Figure 5) or Chl-a fluorescence (Figure 6) by S. mucronata up to 5 individuals (inds.) mL−1. However, Fv/Fm and NPQNSV were significantly affected when S. mucronata was 7.5 inds·mL−1. Therefore, for measuring the photophysiology of Colacium sp. during the attached stage, we chose S. mucronata with the higher burden of Colacium sp. in order to reach sufficient Colacium sp. abundance (>50 cells·mL−1) and a low number of S. mucronata (≤5 inds·mL−1) in the cuvette.

Table 3 shows seasonal variation in photophysiology of Colacium sp. during the attached stage. Although sampling temperature varied, their photophysiology remained relatively constant. σPSII varied from 3.42 nm2 to 3.76 nm2 (mean 3.60 nm2), Fv/Fm varied from 0.52 to 0.60 (mean 0.55), and NPQNSV varied from 0.66 to 0.85 (mean 0.82). To validate these results, we further investigated variations in Colacium sp. photophysiology during the planktonic stage for the stationary phase in the AF-6 medium (Table 4). Mean Fv/Fm and NPQNSV for the attached stage were similar to those of the planktonic stage when incubated in AF-6 medium.

To determine the effect of Ca and Mn on Colacium sp. photophysiology in both the attached and planktonic stages, we performed Ca and Mn enrichment experiments. Samples were taken from the reed area of Lake Biwa on May 7, 2021. For the attached stage of Colacium sp. under dark conditions, there was no significant difference in photophysiological parameters among treatments, except for NPQNSV between Mn and Ca treatments at 3 h, where Ca < Mn (Figure 7A,C,E). Further, σPSII, Fq′/Fm, and NPQNSV responses to increasing light during the attached stage showed no clear differences among treatments (Figure 8A,C,E and Figure 9A,C,E). However, NPQNSV tended to be lower in the Ca treatment than the control at a low light intensity at 21 h (11 and 25 µmol photon·m−2·s−1, Figure 9E). For the planktonic stage, σPSII was significantly lower in the Mn than Ca treatment at 3 h (Figure 7B). Fq′/Fm was significantly higher, but NPQNSV was lower in the Mn treatment than control at 21 h (Figure 7D,F). Under increasing light, Mn tended to decrease σPSII and increase Fq′/Fmduring the planktonic stage, compared to the control at 3 h (Figure 8D). Similarly, Mn significantly reduced NPQNSV during the planktonic stage compared to the control at 21 h (Figure 9F). Similar to the attached stage, calcium slightly improved NPQNSV for the planktonic stage under increasing light (Figure 9F). However, Ca decreased Fq′/Fm and increased NPQNSV for the planktonic stage compared to Mn treatment under 44-200 µmol photon·m−2·s−1 at 3 h (Figure 8D,F).

Figure 1
Figure 1: Colacium sp. and substance organism Scapholeberis mucronata. (A) Infected S. mucronata. (B) Infected S. mucronata fixed with glutaraldehyde. (C) Attached Colacium cells on living S. mucronata. (D) Attached Colacium cells on the molted carapace. (E, F) Colacium sp. of planktonic (palmella) stage. (G) Non-infected S. mucronata. Arrows indicate Colacium cells. Scale bars: 200 µm (A, B, and G), 10 µm (C, E, and F), and 100 µm (D). Please click here to view a larger version of this figure.

Figure 2
Figure 2: Washing zooplankton by pipetting under filtered lake water (FLW). Please click here to view a larger version of this figure.

Figure 3
Figure 3: Act2Run software user interface. (A) Exposure time to each actinic light step; (B) Number of steps and photon flux of actinic light; (C) Combination of excitation wavelength; (D) Saturation and relaxation flashlet sequence; (E) Frequencies and intensities of the water jacket and sample mixing pumps; (F) Photon flux of excitation flash at 444 (denoted as 450 here), 512 (530), and 633 nm (624), photomultiplier tube (PMT) voltage, and replicates and interval of sequence. Please click here to view a larger version of this figure.

Figure 4
Figure 4: Fluorescence reading of the algal sample on Act2Run software. The red and blue lines indicate raw fluorescence signal by a sequence of flashlet and curve fitting in both saturation and relaxation phases, respectively. See Kolber et al.2 for more details. Please click here to view a larger version of this figure.

Figure 5
Figure 5: The effect of S. mucronata density on baseline fluorescence. The small dots represent replicates (n = 3). The results of the ANOVA test are also shown. Please click here to view a larger version of this figure.

Figure 6
Figure 6: The effects of S. mucronata densities on (A) FO (B) σPSII, (C) Fv/Fm, and (D) NPQNSV for Colacium sp. during the planktonic stage. The small dots represent replicates (n = 3). Colacium sp. was cultured in AF-6 medium. The results of ANOVA and Tukey post-hoc test are also presented. Please click here to view a larger version of this figure.

Figure 7
Figure 7: Responses of (A,B) absorption cross-section, (C,D) PSII photochemistry, and (E,F) non-photochemical quenching of (A,C,E) attached stage and (B,D,F) planktonic stage of Colacium sp. at 3 h and 21 h after Ca and Mn addition. The small dots represent replicates (n = 4). The results of ANOVA and Tukey post-hoc test are also presented. * p < 0.05. Please click here to view a larger version of this figure.

Figure 8
Figure 8: Rapid-light responses of (A, B) absorption cross-section, (C,D) PSII photochemistry, and (E,F) non-photochemical quenching of Colacium sp. in attached and planktonic stages to stepwise light protocol at 3 h after Ca and Mn addition. C, control; Ca, 200 µM Ca; Mn, 40 µM Mn. Significant differences between (a) C and Ca, (b) C and Mn, and (c) Ca and Mn at each PAR flux, with a significance level of p < 0.05 shown in each panel. Error bar, Mean SD (n = 4). Please click here to view a larger version of this figure.

Figure 9
Figure 9: Rapid-light responses of (A,B) absorption cross-section, (C,D) PSII photochemistry, and (E,F) non-photochemical quenching of Colacium sp. in attached and planktonic stages to stepwise light protocol at 21 h after Ca and Mn addition. C, control; Ca, 200 µM Ca; Mn, 40 µM Mn. Significant differences between (a) C and Ca, (b) C and Mn, and (c) Ca and Mn at each PAR flux, with a significance level of p < 0.05 shown in each panel. Error bar, Mean SD (n = 4). Please click here to view a larger version of this figure.

Term Definition Units
Baseline fluorescence  Fo value without Chl-a fluorescence
F' Fluorescence at zeroth flashlet of a single turnover measurement when C>0
Fo (ʹ) Minimum PSII Fluorescence yield (under background light) at zeroth flashlet
Fv (ʹ) Fm(ʹ) − Fo(ʹ)
Fm (ʹ) Maximum PSII Fluorescence yield (under background light)
Fv/Fm Maximum PSII photochemical efficiency under dark
Fqʹ/Fmʹ Maximum PSII photochemical efficiency under background light, (FmʹF)/(Fmʹ)
NPQNSV Normalized Stern-Volmer quenching, FOʹ/(Fmʹ FOʹ)
[RCII] Concentration of reaction center
PSII (ʹ) Probability of an RCII being closed during the first flashlet of a single turnover saturation phase (under background light)
σPSII (ʹ) Functional absorption cross section of PSII for excitation flashlets (under background light) nm2

Table 1: Terms used in this protocol.

Component Quantity
NaNO3 140 mg·L1
NH4NO3 22 mg·L1
MgSO4·7H2O 30 mg·L1
KH2PO4 10 mg·L1
K2HPO4 5 mg·L1
CaCl2·2H2O 10 mg·L1
CaCO3 10 mg·L1
Fe-citrate* 2 mg·L1
Citric acid* 2 mg·L1
Biotin 0.002 mg·L1
Vit. B1 0.01 mg·L1
Vit. B6 0.001 mg·L1
Vit. B12 0.001 mg·L1
Trace metals 1 mL·L1
(FeCl3·6H2O) (1.0 mg·mL1)
(MnCl3·4H2O) (0.4 mg·mL1)
(ZnSO4·7H2O) (0.005 mg·mL1)
(CoCl2·6H2O) (0.002 mg·mL1)
(Na2MoO4) (0.004 mg·mL1)
(Na2-EDTA) (7.5 mg·mL−1)

Table 2: Recipe for AF-6 medium. Adjust pH to 6.6. Dissolve Fe-citrate and citric acid in warm H2O separately and add HCl (1 mL·L−1) after mixing both reagents. Contents of trace metals are shown in parenthesis.

Sampling date Sample No. Water temp.
(°C)
S. mucronata density
(inds.·mL1)
Colacium sp. cell density
(inds.·mL1)
σPSII (nm2) Fv/Fm NPQNSV
April 27/2020 No. 1 14.2 4.5 77 3.42 0.60 0.66
SE 0.22 0.01 0.04
May 21/2020 No. 2 19.4 2 282.5 3.62 0.54 0.85
SE 0.16 0.02 0.06
No.3 19.4 2 250.5 3.55 0.56 0.77
SE 0.09 0.01 0.02
No.4 19.4 5 204.5 3.76 0.52 0.94
SE 0.12 0.00 0.02
June 18/2020 No.5 22.4 2.5 474 3.62 0.54 0.85
SE 0.16 0.02 0.06
No.6 22.4 2 410 3.55 0.56 0.77
SE 0.09 0.01 0.02
No.7 22.4 2.5 441 3.76 0.52 0.94
SE 0.12 0.00 0.02
July 20/2020 No. 8 27.5 5 109 3.49 0.58 0.74
SE 0.10 0.00 0.00
Mean 3.60 0.55 0.82
S.D. 0.120349 0.03 0.10

Table 3: Photophysiology of Colacium sp. attached on S. mucronata.

Sampling date Sample No. Medium Growth temperature (°C) σPSII (nm2) Fv/Fm NPQNSV
May 21/2020 No. 1 AF-6 19.4 2.72 0.65 0.53
SE 0.03 0.00 0.01
June 18/2020 No. 2 AF-6 22.4 3.07 0.55 0.84
SE 0.08 0.02 0.07
July 20/2020 No.3 AF-6 27.5 2.90 0.58 0.73
SE 0.06 0.01 0.02
Mean 2.90 0.59 0.70
S.D. 0.18 0.05 0.16

Table 4: Photophysiology of Colacium sp. planktonic stage. Each sample was measured during the stationary phase.

Subscription Required. Please recommend JoVE to your librarian.

Discussion

This protocol demonstrated for the first time that photophysiology of Colacium sp. during the attached stage in a natural environment is comparable to its planktonic stage in AF-6 medium. Additionally, gut contents of starved S. mucronata did not affect baseline and Chl-a fluorescence when density was ≤5 inds·mL−1 (Figure 5 and Figure 6). These results suggest this protocol can measure photophysiology of Colacium sp. during the attached stage without correction under low substrate organism abundance. However, results from steps 3.2.1-3.2.8 showed that the highest S. mucronata abundance affected Fv/Fm and NPQNSV significantly, but not FO and σPSII (Figure 4). Here, it's possible higher organism density exacerbated physical stress on Colacium sp. individuals and subsequently decreased photosynthetic activity. For measurements under a high abundance of substrate organisms or other species, the effects of substrate organism density on the baseline and Chl-a fluorescence requires further attention.

FRRfs have been used to examine the impact of nutrient manipulation on the linear electron flow and non-photochemical quenching of phytoplankton22,56,57. The primary results show that Ca and Mn enrichment differed significantly between Colacium sp. life stages (Figure 7, Figure 8, and Figure 9). Specifically, manganese clearly improved the (maximum) photochemical yield of PSII (Fv/Fand Fq′/Fm) and decreased the heat dissipation (NPQNSV)50 of planktonic stages under dark (Figure 7D,F) and light conditions (Figure 8D,F and Figure 9D,F). These outcomes can stem from reduced antenna size on PSII, σPSII, and σPSII (Figure 7B and Figure 8B), which reduces excess light absorption58,59. Measuring antenna size in addition to energy flow between PSII complexes would allow more precise measurements of the algal response10. This protocol also allows the examination of photosynthesis limitations by other resources. For example, nitrogen and phosphorus limitations have been examined in various phytoplankton communities, but not in epizoic algae, despite predicted effects on Colacium41 and marine epizoic diatoms60,61. In addition to nutrients, the light environment can further influence epizoic algae distribution44.

As shown in Figure 7, Figure 8, and Figure 9, cuvette-type FRRf enables us to simultaneously examine nutrient and light effects without long incubation times and measurement effort. This stepwise light protocol (step 6.1.5) can also draw rapid-light curves of relative electron transport rates (rETR = Fq′/Fm× light) vs. light as an analog for production vs. light curves62. However, although linear electron flow in PSII can be estimated from photophysiological parameters by the FRRf, it is not necessarily analogous to the carbon fixation rate63,64. For estimating carbon-based primary production, electron requirement per CO2 fixation (Фe, C), which can vary temporally and spatially5,48, should be examined when assessing subject communities.

If the plankton net is clogged by debris, prescreen by a larger mesh, such as a 5-mm mesh net, or pick zooplanktons directly from the lake water using a pipette without filtration. It should be noted that some damage might occur to the attached algae even when the filtration is conducted gently using a relatively large (200 µm) mesh size. Although the results show that the standard deviation of the PSII parameters was small (Table 3), and the mean values of the parameters were very similar to those of the cultured planktonic stage (Table 4), sampling without filtration might be ideal.

Another limitation of this study was deriving σPSII. Actinic light and Chl-a fluorescence attenuation can exert a major influence/distortion on the FRRfs, which relies upon optically thin samples for the accurate σPSII determination65. Although we showed that S. mucronata did not affect the σPSII of planktonic Colacium cells, that should be examined related to the σPSII of the attached Colacium cells. Furthermore, a spectral correction factor (SCF) would be needed for the σPSII in situ66 estimation as the excitation wavelength of the ACT2 system (444 nm) differs from the spectral distribution of the in situ light environment. In general, the filter pad technique is used to measure the Chl-a specific absorption spectrum to calculate the SCF. This procedure is necessary to estimate algal primary productivity by the FRRfs. As we could not harvest enough attached Colacium cells through the study period, the Chl-a specific absorption spectrum should be examined in future studies.

Implementing cuvette-type FRRf should depend on substrate size as periphytic algae require a substrate attachment. For example, studies of algae on indestructible substances, such as rocks67, larger organisms26,68, or symbiotic algae, including Symbiodinium associated with hard corals10,69,70, might require the submersible-type FRRf69. Conversely, if the basibiont is small enough to suspend itself in a cuvette, a cuvette-type FRRf might be sufficient in addition to a cuvette-type PAM, such as benthic algae16,17,18. Indeed, recent studies have explored a cuvette-type FRRf for measuring the photophysiology of ice algae24,25. Furthermore, turning on the excitation flash at 512 and 633 nm enables the application to cyanobacteria with different PSII antenna pigments, phycoerythrins and phycocyanins, and thus different absorption peaks with Chl-a71. As current FRRf models incorporating multi-excitation wavelengths are useful tools for examining cyanobacteria photophysiology and productivity7,66,72, these ought to be useful methods for assessing benthic cyanobacteria, if the effects of sample thickness on photophysiological parameters would be improved65. In future studies, FRRfs should be aimed at a wider range of subject organisms to shed further insight on the complex mechanisms of algal photophysiology across various habitats.

Subscription Required. Please recommend JoVE to your librarian.

Disclosures

The authors have no disclosures to declare.

Acknowledgments

The work was supported by the Collaborative Research Fund from Shiga Prefecture entitled "Study on water quality and lake-bottom environment for the protection of the soundness of water environment" under the Japanese Grant for Regional Revitalization and the Environment Research and Technology Development Fund (No. 5-1607) of the Ministry of the Environment, Japan. https://www.kantei.go.jp/jp/singi/tiiki/tiikisaisei/souseikoufukin.html. The authors would like to thank Enago (www.enago.jp) for the English language review.

Materials

Name Company Catalog Number Comments
Acrodisc syringe filter Pall Corporation, Ann Arbor, MI, USA 0.2 μm pore size
Act2Run CTG Ltd., West Molesey, UK
Biotin Wako 023-08711 AF-6 medium
CaCl2·2H2O Wako 031-25031 AF-6 medium
CaCO3 Wako 036-00382 AF-6 medium
Citric acid Wako 036-05522 AF-6 medium
CoCl2·6H2O Wako 036-03682 AF-6 medium
Concentrated Chlorella Recenttec, Tokyo, Japan 20 mg C·mL1 ; store at 4 °C
FastOcean Act2 CTG Ltd., West Molesey, UK
Fe-citrate Wako 093-00952 AF-6 medium
FeCl3·6H2O Wako 091-00872 AF-6 medium
HCLP-880PF Nippon Medical and Chemical Instruments
 Co., Ltd., Osaka, Japan
With LED light bulbs
K2HPO4 Wako 160-04292 AF-6 medium
KH2PO4 Wako 167-04241 AF-6 medium
MgSO4·7H2O Wako 137-00402 AF-6 medium
MnCl3·4H2O Wako 139-00722 AF-6 medium
Na2EDTA Wako 343-01861 AF-6 medium
Na2MoO4 Wako 196-02472 AF-6 medium
NaNO3 Wako 191-02542 AF-6 medium
NH4NO3 Wako 015-03231 AF-6 medium
Plankton Counter Matsunami Glass, Osaka, Japan S6300
Pylex test tube CTG Ltd., West Molesey, UK With rim, 16 x 100 mm
Vit. B1 Wako 203-00851 AF-6 medium
Vit. B12 Wako 226-00343 AF-6 medium
Vit. B6 Wako 165-05401 AF-6 medium
ZnSO4·7H2O Wako 264-00402 AF-6 medium

DOWNLOAD MATERIALS LIST

References

  1. Kolber, Z., Falkowski, P. G. Use of active fluorescence to estimate phytoplankton photosynthesis in situ. Limnology and Oceanography. 38, (8), 1646-1665 (1993).
  2. Kolber, Z. S., Prášil, O., Falkowski, P. G. Measurements of variable chlorophyll fluorescence using fast repetition rate techniques: defining methodology and experimental protocols. Biochimica et Biophysica Acta (BBA) - Bioenergetics. 1367, (1), 88-106 (1998).
  3. Oxborough, K., Moore, C. M., Suggett, D. J., Lawson, T., Chan, H. G., Geider, R. J. Direct estimation of functional PSII reaction center concentration and PSII electron flux on a volume basis: a new approach to the analysis of Fast Repetition Rate fluorometry (FRRf) data. Limnology and Oceanography: Methods. 10, (3), 142-154 (2012).
  4. Smyth, T. J., Pemberton, K. L., Aiken, J., Geider, R. J. A methodology to determine primary production and phytoplankton photosynthetic parameters from fast repetition rate fluorometry. Journal of Plankton Research. 26, (11), 1337-1350 (2004).
  5. Lawrenz, E., et al. Predicting the electron requirement for carbon fixation in seas and oceans. PLoS ONE. 8, (3), 58137 (2013).
  6. Zhu, Y., et al. Relationship between light, community composition and the electron requirement for carbon fixation in natural phytoplankton. Marine Ecology Progress Series. 580, 83-100 (2017).
  7. Schuback, N., Tortell, P. D. Diurnal regulation of photosynthetic light absorption, electron transport and carbon fixation in two contrasting oceanic environments. Biogeosciences. 16, (7), 1381-1399 (2019).
  8. Cosgrove, J., Borowitzka, M. A. Chlorophyll fluorescence terminology: an introduction. Chlorophyll a Fluorescence in Aquatic Sciences: Methods and Applications. Springer. Dordrecht. 1-17 (2010).
  9. McKew, B. A., et al. The trade-off between the light-harvesting and photoprotective functions of fucoxanthin-chlorophyll proteins dominates light acclimation in Emiliania huxleyi (clone CCMP 1516). New Phytologist. 200, (1), 74-85 (2013).
  10. Warner, M. E., Lesser, M. P., Ralph, P. J. Chlorophyll fluorescence in reef building corals. Chlorophyll a Fluorescence in Aquatic Sciences: Methods and Applications. Springer. Dordrecht. 209-222 (2010).
  11. Bhagooli, R., et al. Chlorophyll fluorescence - A tool to assess photosynthetic performance and stress photophysiology in symbiotic marine invertebrates and seaplants. Marine Pollution Bulletin. 165, 112059 (2021).
  12. Zavafer, A., Labeeuw, L., Mancilla, C. Global trends of usage of chlorophyll fluorescence and projections for the next decade. Plant Phenomics. 2020, 6293145 (2020).
  13. Goto, N., Tanaka, Y., Mitamura, O. Relationships between carbon flow through freshwater phytoplankton and environmental factors in Lake Biwa, Japan. Fundamental and Applied Limnology/Archiv für Hydrobiologie. 184, (4), 261-275 (2014).
  14. Napoléon, C., Raimbault, V., Claquin, P. Influence of nutrient stress on the relationships between PAM measurements and carbon incorporation in four phytoplankton species. PLOS ONE. 8, (6), 66423 (2013).
  15. Morris, E. P., Kromkamp, J. C. Influence of temperature on the relationship between oxygen- and fluorescence-based estimates of photosynthetic parameters in a marine benthic diatom (Cylindrotheca closterium). European Journal of Phycology. 38, (2), 133-142 (2003).
  16. Fraga, S., Rodríguez, F., Bravo, I., Zapata, M., Marañón, E. Review of the main ecological features affecting benthic dinoflagellate blooms. Cryptogamie, Algologie. 33, (2), 171-179 (2012).
  17. McMinn, A., et al. Quantum yield of the marine benthic microflora of near-shore coastal Penang, Malaysia. Marine and Freshwater Research. 56, (7), 1047-1053 (2005).
  18. Salleh, S., McMinn, A. The effects of temperature on the photosynthetic parameters and recovery of two temperate benthic microalgae, Amphora cf. coffeaeformis and Cocconeis cf. sublittoralis (Bacillariophyceae). Journal of Phycology. 47, (6), 1413-1424 (2011).
  19. McMinn, A., Pankowskii, A., Ashworth, C., Bhagooli, R., Ralph, P., Ryan, K. In situ net primary productivity and photosynthesis of Antarctic sea ice algal, phytoplankton and benthic algal communities. Marine Biology. 157, (6), 1345-1356 (2010).
  20. Garbary, D. J., Bird, C. J., Kim, K. Y. Sporocladopsis jackii, sp. nov. (Chroolepidaceae, chlorophyta): a new species from eastern Canada and Maine symbiotic with the mud snail, Ilyanassa obsoleta (Gastropoda). Rhodora. 107, (929), 52-68 (2005).
  21. Suggett, D. J., Oxborough, K., Baker, N. R., MacIntyre, H. L., Kana, T. M., Geider, R. J. Fast repetition rate and pulse amplitude modulation chlorophyll a fluorescence measurements for assessment of photosynthetic electron transport in marine phytoplankton. European Journal of Phycology. 38, (4), 371-384 (2003).
  22. Hughes, D. J., et al. Impact of nitrogen availability upon the electron requirement for carbon fixation in Australian coastal phytoplankton communities. Limnology and Oceanography. 63, (5), 1891-1910 (2018).
  23. Melrose, D. C., Oviatt, C. A., O'Reilly, J. E., Berman, M. S. Comparisons of fast repetition rate fluorescence estimated primary production and 14C uptake by phytoplankton. Marine Ecology Progress Series. 311, 37-46 (2006).
  24. Yoshida, K., Seger, A., Kennedy, F., McMinn, A., Suzuki, K. Freezing, melting, and light stress on the photophysiology of ice algae: ex situ incubation of the ice algal diatom Fragilariopsis cylindrus (Bacillariophyceae) using an ice tank. Journal of Phycology. 56, (5), 1323-1338 (2020).
  25. Selz, V., et al. Ice algal communities in the Chukchi and Beaufort Seas in spring and early summer: composition, distribution, and coupling with phytoplankton assemblages. Limnology and Oceanography. 63, (3), 1109-1133 (2018).
  26. Falasco, E., Bo, T., Ghia, D., Gruppuso, L., Bona, F., Fenoglio, S. Diatoms prefer strangers: non-indigenous crayfish host completely different epizoic algal diatom communities from sympatric native species. Biological Invasions. 20, (10), 2767-2776 (2018).
  27. Møhlenberg, F., Kaas, H. Colacium vesiculosum Ehrenberg (Euglenophyceae), infestation of planktonic copepods in the Western Baltic. Ophelia. 31, (2), 125-132 (1990).
  28. Zalocar, Y., Frutos, S. M., Casco, S. L., Forastier, M. E., Vallejos, S. V. Prevalence of Colacium vesiculosum (Colaciales: Euglenophyceae) on planktonic crustaceans in a subtropical shallow lake of Argentina. Revista De Biologia Tropical. 59, (3), 1295-1306 (2011).
  29. Barea-Arco, J., Pérez-Martínez, C., Morales-Baquero, R. Evidence of a mutualistic relationship between an algal epibiont and its host, Daphnia pulicaria. Limnology and Oceanography. 46, (4), 871-881 (2001).
  30. Decaestecker, E., Declerck, S., De Meester, L., Ebert, D. Ecological implications of parasites in natural Daphnia populations. Oecologia. 144, (3), 382-390 (2005).
  31. Allen, Y. C., Stasio, B. T. D., Ramcharan, C. W. Individual and population level consequences of an algal epibiont on Daphnia. Limnology and Oceanography. 38, (3), 592-601 (1993).
  32. Willey, R. L., Cantrell, P. A., Threlkeld, S. T. Epibiotic euglenoid flagellates increase the susceptibility of some zooplankton to fish predation. Limnology and Oceanography. 35, (4), 952-959 (1990).
  33. Green, J. Parasites and epibionts of Cladocera. The Transactions of the Zoological Society of London. 32, (6), 417-515 (1974).
  34. Evans, M. S., Sicko-Goad, L. M., Omair, M. Seasonal occurrence of Tokophrya quadripartita (Suctoria) as epibionts on adult Limnocalanus macrurus (Copepoda: Calanoida) in southeastern Lake Michigan. Transactions of the American Microscopical Society. 98, (1), 102-109 (1979).
  35. Chiavelli, D. A., Mills, E. L., Threlkeld, S. T. Host preference, seasonality, and community interactions of zooplankton epibionts. Limnology and Oceanography. 38, (3), 574-583 (1993).
  36. Willey, R. L., Willey, R. B., Threlkeld, S. T. Planktivore effects on zooplankton epibiont communities: epibiont pigmentation effects. Limnology and Oceanography. 38, (8), 1818-1822 (1993).
  37. Rosowski, J. R., Willey, R. L. Colacium libellae sp. nov. (euglenophyceae), a photosynthetic inhabitant of the larval damselfly rectum. Journal of Phycology. 11, (3), 310-315 (1975).
  38. Willey, R. L., Threlkeld, S. T. Organization of crustacean epizoan communities in a chain of subalpine ponds. Limnology and Oceanography. 38, (3), 623-627 (1993).
  39. Al-Dhaheri, R. S., Willey, R. L. Colonization and reproduction of the epibiotic flagellate Colacium vesiculosum (euglenophyceae) on Daphnia pulex. Journal of Phycology. 32, (5), 770-774 (1996).
  40. Rosowski, J. R. Photosynthetic euglenoids. Freshwater Algae of North America. Elsevier Science. USA. 383-422 (2003).
  41. Rosowski, J. R., Kugrens, P. Observations on the euglenoid Colacium with special reference to the formation and morphology of attachment material. Journal of Phycology. 9, (4), 370-383 (1973).
  42. Salmaso, N., Tolotti, M. Other phytoflagellates and groups of lesser importance. Encyclopedia of Inland Waters. Academic Press. 174-183 (2009).
  43. Threlkeld, S. T., Chiavelli, D. A., Willey, R. L. The organization of zooplankton epibiont communities. Trends in Ecology & Evolution. 8, (9), 317-321 (1993).
  44. Bertolo, A., Rodríguez, M. A., Lacroix, G. Control mechanisms of photosynthetic epibionts on zooplankton: an experimental approach. Ecosphere. 6, (11), (2015).
  45. Pringsheim, E. G. Notiz über Colacium (Euglenaceae). Österreichische Botanische Zeitschrift. 100, (3), 270-275 (1953).
  46. Wołowski, K., Duangjan, K., Peerapornpisal, Y. Colacium minimum (Euglenophyta), a new epiphytic species for Asia. Polish Botanical Journal. 60, (2), 179-185 (2015).
  47. Martin, J. H., Knauer, G. A. The elemental composition of plankton. Geochimica et Cosmochimica Acta. 37, (7), 1639-1653 (1973).
  48. Kazama, T., Hayakawa, K., Kuwahara, V. S., Shimotori, K., Imai, A., Komatsu, K. Development of photosynthetic carbon fixation model using multi-excitation wavelength fast repetition rate fluorometry in Lake Biwa. PLOS ONE. 16, (2), 0238013 (2021).
  49. Chesney, T., Sastri, A. R., Beisner, B. E., Nandini, S., Sarma, S. S. S., Juneau, P. Application of fluorometry (Phyto-PAM) for assessing food selection by cladocerans. Hydrobiologia. 829, (1), 133-142 (2019).
  50. Wang, Q., Yang, S., Wan, S., Li, X. The significance of calcium in photosynthesis. International Journal of Molecular Sciences. 20, (6), 1353 (2019).
  51. Dau, H., Haumann, M. Eight steps preceding O-O bond formation in oxygenic photosynthesis-A basic reaction cycle of the photosystem II manganese complex. Biochimica et Biophysica Acta (BBA) - Bioenergetics. 1767, (6), 472-483 (2007).
  52. Suthers, I., Bowling, L., Kobayashi, T., Rissik, D. Sampling methods for plankton. Plankton: A guide to their ecology and monitoring for water quality. CSIRO Publishing. Melbourne. 63-90 (2019).
  53. Błędzki, L. A., Rybak, J. I. Freshwater Crustacean Zooplankton of Europe: Cladocera & Copepoda (Calanoida, Cyclopoida) Key to species identification, with notes on ecology, distribution, methods and introduction to data analysis. Springer. Switzerland. (2016).
  54. Kato, S. Laboratory culture and morphology of Colacium vesiculosum Ehrb. (Euglenophyceae). Japanese Journal of Phycology (Sorui). 30, 63-67 (1982).
  55. Serôdio, J., Campbell, D. A. Photoinhibition in optically thick samples: Effects of light attenuation on chlorophyll fluorescence-based parameters. Journal of Theoretical Biology. 513, 110580 (2021).
  56. Sylvan, J. B., Quigg, A., Tozzi, S., Ammerman, J. W. Eutrophication-induced phosphorus limitation in the Mississippi River plume: evidence from fast repetition rate fluorometry. Limnology and Oceanography. 52, (6), 2679-2685 (2007).
  57. Browning, T. J., et al. P. Nutrient regulation of late spring phytoplankton blooms in the midlatitude North Atlantic. Limnology and Oceanography. 65, (6), 1136-1148 (2020).
  58. Pausch, F., Bischof, K., Trimborn, S. Iron and manganese co-limit growth of the Southern Ocean diatom Chaetoceros debilis. PLOS ONE. 14, (9), 0221959 (2019).
  59. Ferroni, L., Baldisserotto, C., Fasulo, M. P., Pagnoni, A., Pancaldi, S. Adaptive modifications of the photosynthetic apparatus in Euglena gracilis Klebs exposed to manganese excess. Protoplasma. 224, (3), 167-177 (2004).
  60. Gaiser, E. E., Bachmann, R. W. Seasonality, substrate pereference and attachment sites of epizoic diatoms on cladoceran zooplankton. Journal of Plankton Research. 16, (1), 53-68 (1994).
  61. Totti, C., et al. The diversity of epizoic diatoms: relationships between diatoms and marine invertebrates. The Diversity of Epizoic Diatoms. 16, 323-343 (2011).
  62. Perkins, M., Effler, S. W., Strait, C. M. Phytoplankton absorption and the chlorophyll a-specific absorption coefficient in dynamic Onondaga Lake. Inland Waters. 4, (2), 133-146 (2014).
  63. Kromkamp, J., Capuzzo, E., Philippart, C. J. M. Measuring phytoplankton primary production: review of existing methodologies and suggestions for a common approach. EcApRHA Deliverable WP 3.2. 28, (2017).
  64. Hughes, D., et al. Roadmaps and detours: active chlorophyll-a assessments of primary productivity across marine and freshwater systems. Environmental Science & Technology. 52, (21), 12039-12054 (2018).
  65. Perkins, R. G., et al. The application of variable chlorophyll fluorescence to microphytobenthic biofilms. Chlorophyll a Fluorescence in Aquatic Sciences: Methods and Applications. 4, Springer. Dordrecht. 237-275 (2010).
  66. Schuback, N., Flecken, M., Maldonado, M. T., Tortell, P. D. Diurnal variation in the coupling of photosynthetic electron transport and carbon fixation in iron-limited phytoplankton in the NE subarctic Pacific. Biogeosciences. 13, (4), 1019-1035 (2016).
  67. Schreiber, U., Gademann, R., Ralph, P. J., Larkum, A. W. D. Assessment of photosynthetic performance of Prochloron in Lissoclinum patella in hospite by chlorophyll fluorescence measurements. Plant and Cell Physiology. 38, (8), 945-951 (1997).
  68. Garbary, D. J., Miller, A. G., Scrosati, R. A. Ascophyllum nodosum and its symbionts: XI. The epiphyte Vertebrata lanosa performs better photosynthetically when attached to Ascophyllum than when alone. Algae. 29, (4), 321-331 (2014).
  69. Gorbunov, M. Y., Kolber, Z. S., Lesser, M. P., Falkowski, P. G. Photosynthesis and photoprotection in symbiotic corals. Limnology and Oceanography. 46, (1), 75-85 (2001).
  70. Yellowlees, D., Warner, M. Photosynthesis in symbiotic algae. Photosynthesis in Algae. 14, Springer. Dordrecht. 437-455 (2003).
  71. Wojtasiewicz, B., Stoń-Egiert, J. Bio-optical characterization of selected cyanobacteria strains present in marine and freshwater ecosystems. Journal of Applied Phycology. 28, (4), 2299-2314 (2016).
  72. Aardema, H. M., Rijkeboer, M., Lefebvre, A., Veen, A., Kromkamp, J. C. High-resolution underway measurements of phytoplankton photosynthesis and abundance as an innovative addition to water quality monitoring programs. Ocean Science. 15, (5), 1267-1285 (2019).
This article has been published
Video Coming Soon
PDF DOI DOWNLOAD MATERIALS LIST

Cite this Article

Kazama, T., Hayakawa, K., Shimotori, K., Imai, A. Measuring Photophysiology of Attached Stage of Colacium sp. by a Cuvette-Type Fast Repetition Rate Fluorometer. J. Vis. Exp. (177), e63108, doi:10.3791/63108 (2021).More

Kazama, T., Hayakawa, K., Shimotori, K., Imai, A. Measuring Photophysiology of Attached Stage of Colacium sp. by a Cuvette-Type Fast Repetition Rate Fluorometer. J. Vis. Exp. (177), e63108, doi:10.3791/63108 (2021).

Less
Copy Citation Download Citation Reprints and Permissions
View Video

Get cutting-edge science videos from JoVE sent straight to your inbox every month.

Waiting X
Simple Hit Counter