Nov 13, 2021

Public workspaceMeasuring Photophysiology of Attached Stages of Colacium sp. by a Cuvette-Type Fast Repetition Rate Fluorometer V.2

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  • Takehiro Kazama1,2,
  • Kazuhide Hayakawa3,
  • Koichi Shimotori1,4,
  • Akio Imai1
  • 1Lake Biwa Branch Office, National Institute for Environmental Studies, Otsu, Shiga 520-0022, Japan;
  • 2Center for Regional Environmental Research, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japa;
  • 3Lake Biwa Environmental Research Institute, Otsu, Shiga 520-0022, Japan;
  • 4Center for Regional Environmental Research, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan
  • FRRf in Lake Biwa
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Document Citation: Takehiro Kazama, Kazuhide Hayakawa, Koichi Shimotori, Akio Imai 2021. Measuring Photophysiology of Attached Stages of Colacium sp. by a Cuvette-Type Fast Repetition Rate Fluorometer. protocols.io https://protocols.io/view/measuring-photophysiology-of-attached-stages-of-co-bz26p8heVersion created by kazama
License: This is an open access document distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Created: November 13, 2021
Last Modified: November 13, 2021
Document Integer ID: 55102
Keywords: bench-top FRRf, Colacium sp., epibiont, epizoic algae, Lake Biwa, photophysiology
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, we describe 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 background fluorescence and σPSII, Fv/Fm, Fq′/Fm′, and NPQNSV of planktonic Colacium sp. To validate our 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 can determine effects of Ca and 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.
Measuring Photophysiology of Attached Stage of Colacium sp. by a Cuvette- Type Fast Repetition Rate Fluorometer
Last revision: 2021-07-16

AUTHORS AND AFFILIATIONS:
Takehiro Kazama1,2,3, Kazuhide Hayakawa4, Koichi Shimotori1,2, Akio Imai1

1Lake Biwa Branch Office, National Institute for Environmental Studies, Otsu, Shiga 520-0022, Japan
2Center for Regional Environmental Research, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan
3Graduate School of Human Development and Environment, Kobe University, Kobe, Hyogo 657-0011, Japan
4Lake Biwa Environmental Research Institute, Otsu, Shiga 520-0022, Japan

Email: kazama303@gmail.com (TK)

Keywords: bench-top FRRf, Colacium sp., epibiont, epizoic algae, Lake Biwa, photophysiology

Published version: https://dx.doi.org/10.3791/63108 (Journal of Visualized Experiments)

SUMMARY:
Fast repetition rate fluorometer (FRRf) is a beneficial method for measuring photosystem II photophysiology and primary productivity. Here we describe how to measure PSII photophysiology of epizoic alga, Colacium sp. on substrate zooplankton using cuvette-type FRRf.


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 photophysiology1,4 and estimating primary productivity1–3, which enables monitoring phytoplankton PSII photophysiology, as well as primary productivity, across wide spatial and temporal scales5–7. FRRf can simultaneously measure absorption cross section of PSII (σPSII), concentration of reaction center ([RCII]), maximum photochemical efficiency (Fv/Fm), effective photochemical efficiency (Fq′/Fm′), and non-photochemical quenching (NPQNSV) (Table 1). Generally, Fv/Fm and Fq′/Fm′ are defined as PSII activity8, while NPQNSVis 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 NPQNSVby simultaneously measuring recovery kinetics of Fv/Fm, Fq′/Fm′, NPQNSV, and σPSII10. To date, two types of FRRf instruments are commercially available, the submersible-type and cuvette-type. The submersible-type FRRf enables in situ measurements in oceans and lakes, while the cuvette-type FRRf is suitable for measuring small sample volume.

Given the development of PAM fluorometers, including the cuvette-type, for a broad range of subjects11, PAM fluorometers are still more common than the FRRf 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 phytoplankton13–15, benthic microalgae16–18, ice algae19, and epizoic algae20, while the cuvette-type FRRf has been applied primarily to phytoplankton21–23 and a limited number of ice algae species24,25. 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 algae 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 predators32–36. Therefore, exploring the environmental and biological factors controlling epibionts dynamics in zooplankton communities is crucial.

Among epizoic algae, Colacium Ehrenberg 1834 (Euglenophyta) is a common, freshwater, algal group 32,37–39 with various life stages, including an attached stage (Figure 1A–D), non-motile planktonic stage (Figure 1E,F), and motile planktonic stage40,41. In the non-motile planktonic stage, cells live as single-cell plankton, an aggregated colony, or as a one-layer sheet colony, which are covered by mucilage42. In the attached stage, Colacium sp.use mucilage excreted from 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 evolutionally trait for gathering resources, such as light44 and trace elements41,45,46, or as a dispersion strategy27, there is little experimental evidence37,41,44 and the key attachment mechanisms are largely unknown. For example, Rosowski and Kugrens expected that Colacium obtains Mn from substrate copepods41, which concentrated in the exoskeleton47.

Here, we describe how to measure PSII photophysiology of planktonic algae and its application method for targeting attached algae (attaching to zooplankton) with Colacium sp. cells using the cuvette-type FRRf. Since finding the planktonic stage ofColaciumsp. in natural environments is difficult, we collected their attached stage for our experiments. Among the many substrate organisms,Scapholeberis mucronata O.F. Müller 1776 (Branchiopoda, Daphniidae; Figure 1A,B,G) is one of the simplest organisms 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 Colaciumsp. attached on S. mucronata as a case study of the Colacium-basibiont system. Further, we applied this protocol to clarify the attaching mechanism of Colacium sp. and determine the effects of two metals, calcium (Ca) and manganese (Mn) on the photophysiology of both planktonic and attached stages. Calcium plays multiple, key roles in photosynthetic pathways48 and both metals are required to construct oxygen-evolving complexes of PSII49. Because calcium and manganese are highly concentrated in the carapace of crustacean zooplankton47, we hypothesize that Colaciumsp. photophysiology may 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.

PROTOCOL:
1. Sampling
  1. Collect lake waterfrom surface by bucket. To target Colacium sp.attached to S. mucronata (Figure 1A–C), filter 0.5 to 10 L of lake water using a 100 μm nylon mesh net. NOTE: S. mucronata often densely aggregate in shallow, eutrophic, muddy water, such as among reed (phragmites) areas.
  2. Store concentrated samples in 500 mL plastic bottles with some lake water. Keep in dark conditions.
  3. In the laboratory, pour the sample water into a 500 mL beaker and allow to settle for a few minutes.
  4. Filter lake water through a 0.2 μm pore-size filter.
  5. Pick up S. mucronata individuals by pipette. Transfer them into a drop of 0.2 μm filtered lake water (hereafter, FLW) on a slide glass. NOTE: S. mucronata may swim to the surface or attach to the beaker wall.
  6. Check S. mucronata under light microscopy.
  7. Wash S. mucronata individuals using FLW (3 drops or more) to prevent contamination from other organisms (Figure 2).
  8. Keep S. mucronata atan in situ temperature in a growth chamber.

2. Effects of S. mucronata on baseline fluorescence
2. 1. S. mucronata cultivation
  1. Pick up S. mucronata individuals by pipette under an optical microscope and wash using FLW, as in step 1.7.
  2. Pour aerated tap water into a glass jar.
  3. Feed Chlorella (1 mg C L−1) and maintain at 20° under dim light in a growth chamber.
  4. After approximately 14 days, inoculate a sub-culture with clean aerated tap water to keep the medium fresh.

2. 2. FRRf measurements
  1. To examine the effects of zooplankton individuals on baseline fluorescence, prepare adult S. mucronata (body size 400–650 μm) without any attached organisms. To avoid fluorescence from the gut contents, individuals must be starved in FLW at 20° for at least 90 mins.
  2. Pour 1.5 mL of FLW into a cuvette. Transfer 0, 1, 5, and 10 S. mucronata individuals into the cuvette and add FLW to bring the sample up to 2 mL.
  3. Acclimate under low light (1–10 μmol photon m−2 s−1) at20° for 15 mins before FRRf measurement.
  4. Set the operation software to apply a ST method by blue excitation wavelength50,51. Measurements should be repeated >3 times per sample. NOTE: Check the RσPSIIvalue is within the optimal range (0.03–0.08)52.

3. Effects of substrate organism on Chl-a fluorescence
3. 1. Colacium sp. cultivation
  1. Prepare FLW and AF-6 medium53 for cultivation (Table 2).
  2. Keep sampled Colacium sp. on S. mucronata at in situ temperature in a growth chamber.
  3. Pick up Colacium sp. with molted carapace (Figure 1D) by pipette under an optical microscope. Wash them with FLW, as in step 1.7.
  4. Aseptically inoculate Colacium sp. and AF-6 medium ina 10 mL glass tube on a clean bench.
  5. Maintain 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.

3. 2. FRRf measurements
  1. To examine the effects of zooplankton individuals on Chl-a fluorescence from Colaciumsp., prepare adult S. mucronata (body size 400–650 μm) without any attached organisms. To avoid fluorescence from the gut contents, individuals must be starved in FLW for at least 90 mins.
  2. Set up a cuvette-type fast repetition rate fluorometer (FRRf).
  3. 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.
  4. Acclimate under low light (1–10 μmol photon m−2 s−1) at20° for 15 mins before taking the FRRf measurement.
  5. Set the operation software to apply a ST method by blue excitation wavelength50,51. Measurement should be repeated >3 times per sample. NOTE: Maintain samples at incubation temperature during measurements
  6. To correct baseline fluorescence22, filtrate culture medium using a 0.2 µm pore-size filter and measure fluorescence. Subtract FOof the baseline sample from FO and Fm of Colacium sp., or calculate using the operation software.

4. Photophysiology of Colacium sp. (attached stage)
  1. Isolate S. mucronata individuals with Colacium sp. by pipette under an optical microscope.
  2. Wash S. mucronata using FLW, as in step 1.7.
  3. Transfer S. mucronata into a 100 mL of FLW. For starvation, keep under dark conditions at in situ temperature for 90 mins.
  4. Pour 1.5 mL of FLW into a cuvette.
  5. Transfer ~10 S. mucronata individuals with Colaciumsp. 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 mins. Measure Chl-a fluorescence as in step 3.2.5–3.2.6.
  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 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 Colaciumsp. and pour into a cuvette.
  3. Acclimate under low light (1–10 μmol photon m−2 s−1) at in situ temperature for 15 mins. Measure Chl-a fluorescence as in step 3.2.5–3.2.6.

6. Effects of Ca and Mn addition on photophysiology of Colacium sp.
6.1. Effects on attached stage
  1. Isolate S. mucronata individuals with Colacium sp. by pipette under an optical microscope. Wash using FLW, as in step 1.7.
  2. Transfer six individuals into 12 glass beakers with 30 mL of FLW. Each beaker must contain >100 Colacium sp. cells.
  3. Add 200 μmol L−1 CaCl2·H2O (Ca treatment), 40 μmol L−1 MnCl4 (Mn treatment), or MiliQ water (control) to each beaker. Incubate samples under 200 μmol photon m−2 s−1 at in situ temperature in a growth chamber.
  4. At 3 and 21 h, transfer all individuals and molted skins into a cuvette.
  5. After 15 min of dark acclimation, measure Chl-a fluorescence of each sample as in steps 3.2.5–3.2.6. To examine the rapid response to increasing light, expose samples to 20 s periods of 8 actinic lights increasing stepwise in intensity from 0 to 200 μmol photon m−2 s−1 after 30 s measurement in dark conditions. NOTE: Verify RσPSII and RσPSII‘ values are within the optimal range (0.03–0.08)52

6. 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 cultured Colacium sp. into FLW and acclimate at in situ temperature less than 200 μmol photon m−2 s−1 for three days.
  3. Transfer 1 mL of acclimated samples into three glass vials with 10 mL of FLW.
  4. Add 200 μmol L−1 CaCl2·H2O (Ca treatment), 40 μmol L−1MnCl3(Mn treatment), or MiliQ water (control) to vials. Incubate 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 step 6.1.5.

REPRESENTATIVE RESULTS:
There was no significant effect of background fluorescence (Figure 3) or Chl-a fluorescence (Figure 4) by S. mucronata up to 10 inds. [2 mL]−1. However, Fv/Fm and NPQNSVwere significantly affected when S. mucronata was 15 inds. [2 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 (>100 cells [2 mL]−1) and a low number of S. mucronata (≤10 inds. [2 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 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 Colaciumsp. 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 Colaciumsp. 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 5A,C,E). Further, σPSII′, Fq′/Fm′, and NPQNSV responses to increasing light during the attached stage showed no clear differences among treatments (Figure 6A,C,E and Figure 7A,C,E). However, NPQNSV tended to be lower in the Ca treatment than the control at low light intensity at 21 h (11 and 25μmol photon m−2 s−1, Figure 7E). For the planktonic stage, σPSII was significantly lower in the Mn than Ca treatment at 3 h (Figure 5B). Fq′/Fm′ was significantly higher, but NPQNSV was lower in the Mn treatment than control at 21 h (Figure 5D,F). Under increasing light, Mn tended to decrease σPSII and increase Fq′/Fm′ during the planktonic stage, compared to the control at 3 h (Figure 6D). Similarly, Mn significantly reduced NPQNSV during the planktonic stage compared to the control at 21 h (Figure 7F). Similar to the attached stage, calcium slightly improved NPQNSV for the planktonic stage under increasing light (Figure 7F). 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 6D,F).

FIGURE AND TABLE LEGENDS:

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 molted carapace. (E, F)Colacium sp. of planktonic (palmella) stage. (G) Non-infected S. mucronata. Arrows indicate Colacium cells.

Figure 2: Washing zooplankton by pipetting under filtered lake water (FLW).

Figure 3: The effect of S. mucronata density on background fluorescence.
Small dots represent replicates. The results of ANOVA test are also shown.

Figure 4: The effects of S. mucronata densities on (A), (B) σPSII, (C) Fv/Fm, and (D) NPQNSV for Colacium sp. during the planktonic stage.
Small dots represent replicates. Colacium sp. was cultured in AF-6 medium. The results of ANOVA and Tukey post-hoc test are also presented.

Figure 5: 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.
Small dots represent replicates. The results of ANOVA and Tukey post-hoc test are also presented. *, p < 0.05.

Figure 6: 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.

Figure 7: 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.

ABC
TermDefinitionUnits
F'Fluorescence at zeroth flashlet of a single turnover measurement when C>0
Fo (')Minimum PSII Fluorescence yield (under background light)
Fv (')Fm(') − Fo(')
Fm (')Maximum PSII Fluorescence yield (under background light)
Fv/FmMaximum PSII photochemical efficiency under dark
Fq'/Fm'Maximum PSII photochemical efficiency under background light, (Fm' − F)/(Fm’)
NPQNSVNormalized Stern-Volmer quenching
[RCII]Concentration of reaction center
RσPSII (')Probability of an RCII being closed during the first flashlet of a single turnover saturation phase (under background light)
σPSII (')Functional absorption cross section of PSII for excitation flashlets (under background light)nm2

Table 1: Terms used in this protocol.


AB
Component Quantity
NaNO3140 mg L−1
NH4NO322 mg L−1
MgSO4·7H2O30 mg L−1
KH2PO410 mg L−1
K2HPO45 mg L−1
CaCl2·2H2O10 mg L−1
CaCo310 mg L−1
Fe-citrate*2 mg L−1
Citric acid*2 mg L−1
Biotin0.002 mg L−1
Vit. B10.01 mg L−1
Vit. B60.001 mg L−1
Vit. B120.001 mg L−1
Trace metals1 mL L−1
(FeCl3·6H2O)(1 mg L−1)
(MnCl3·4H2O)(0.4 mg L−1)
(ZnSO4·7H2O)(0.005 mg L−1)
(CoCl2·6H2O)(0.002 mg L−1)
(Na2MoO4)(0.004 mg L−1)
(Na2-EDTA)(7.5 mg L−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 1 mL HCl L−1 after mixing both reagents.Contents of trace metals are shown in parenthesis.

ABCDEFGH
Sampling date Sample No. Water temperature (°C)S. mucronata density (inds. [2 mL]−1)Colacium sp. cell density (cells [2 mL] −1)σPSII (nm2)Fv/Fm NPQNSV
April 27/2020 No. 1 14.2 9 154 3.42 0.60 0.66
SE 0.22 0.01 0.04
May 21/2020 No. 2 19.4 4 565 3.62 0.54 0.85
SE 0.16 0.02 0.06
No.3 19.4 4 501 3.55 0.56 0.77
SE 0.09 0.01 0.02
No.4 19.4 10 409 3.76 0.52 0.94
SE 0.12 0.00 0.02
June 18/2020 No.5 22.4 5 948 3.62 0.54 0.85
SE 0.16 0.02 0.06
No.6 22.4 4 820 3.55 0.56 0.77
SE 0.09 0.01 0.02
No.7 22.4 5 882 3.76 0.52 0.94
SE 0.12 0.00 0.02
July 20/2020 No. 8 27.5 10 218 3.49 0.58 0.74
SE 0.10 0.00 0.00
Mean 3.60 0.55 0.82

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

ABCDEFG
Sampling dateSample No. Medium Growth temperature (°C)σPSII (nm2) Fv/FmNPQNSV
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

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



DISCUSSION:
This protocol demonstrated for the first time that photophysiology of Colacium sp. during the attached stage in natural environment is comparable to its planktonic stage in AF-6 medium. Additionally, the effects of a substrate organism of fluorescence was negligible when density was ≤10 inds. [2 mL]−1. 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 to 3.2.6 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 background and Chl-a fluorescence requires further attention.

FRRf have been used to examine the impacts of nutrient manipulation on linear electron flow and non-photochemical quenching of phytoplankton22,54,55. Our primary results show Ca and Mn enrichment differed significantly between Colacium sp. life stages (Figure 5–7). Specifically, manganese clearly improved linear electron flow (Fv/Fmand Fq′/Fm′)and decreased heat dissipation (NPQNSV)48 of planktonic stages under dark (Figure 5D,F) and light conditions (Figure 6D,F and Figure 7D,F). These outcomes can stem from reduced antenna size on PSII, σPSII,and σPSII′ (Figure 5B and Figure 6B), which reduces excess light absorption56,57. Measuring antenna size in addition to energy flow between PSII complexes will allow more precise measurements of algal response10. Our protocol can also examine photosynthesis limitation by other resources. For example, nitrogen and phosphorus limitation have been examined in various phytoplankton communities, but not in epizoic algae, despite predicted effects on Colacium41 and marine epizoic diatoms58,59. In addition to nutrients, the light environment can further influence epizoic algae distribution44.

As shown in Figure 5–7, 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 curves60. However, although linear electron flow in PSII can be estimated from photophysiological parameters by FRRf, it is not necessarily analogous to carbon fixation rate61,62. For estimating production rates based on carbon levels, electron requirement per CO2 fixation (Фe, C), which can vary both temporally and spatially5,52, is recommended when assessing subject communities. Another limitation of our study was deriving σPSIIto represent background light. Because the wavelength of excitation light for FRRf varies, a spectral correction factor should be calculated from a Chl-a specific absorption spectrum of algae, as well as spectral distribution of background light63.

Implementing cuvette-type FRRf should depend on substrate size because periphytic algae require a substrate attachment. For example, studies of algae on indestructible substances, such as rocks64, larger organisms26,65, or symbiotic algae, including Symbiodinium associated with hard corals10,66,67, may require the submersible-type FRRf66. Conversely, if the basibiont is small enough to suspend itself in a cuvette, a cuvette-type FRRf may be sufficient in addition to a cuvette-type PAM, such as benthic algae16–18. Indeed, recent studies have explored a cuvette-type FRRf for measuring photophysiology of ice algae24,25. Because current models of cuvette-type FRRf incorporating multi-excitation wavelengths are useful tools for examining cyanobacteria photophysiology and productivity7,63,68, these ought to be useful methods for assessing benthic cyanobacteria. In future studies, FRRf should be aimed at a wider range of subject organisms to shed further insight on the complex mechanisms of algal photophysiology across various habitats.

ACKNOWLEDGMENTS:
The work was supported by the Collaborative Research Fund from Shiga Prefecture
entitled “Study on water quality and lake-bottom environment for 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.

DISCLOSURES:
The authors have nothing to disclosure.

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