The work was supported by the Collaborative Research Fund from Shiga Prefecture entitled &#34;Study on water quality and lake-bottom environment for the protection of the soundness of water environment&#34; 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.&#160;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.

1. Sampling
<ol>
	<li>Collect lake water&#160;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 &#181;m nylon mesh net52. 
	NOTE: S. mucronata often densely aggregate in shallow, eutrophic, muddy water, such as among reed (phragmites) areas.</li>
	<li>Store the concentrated samples in 500 mL plastic bottles with 350 mL...

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 &#8804;5 inds&#183;mL&#8722;1 (Figure 5 and Figure 6). These results suggest this protocol can measure photophysiology of <em...

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 (&#963;PSII) absorption cross section, reaction center ([RCII]) concentration, maximum photochemical efficiency (Fv/Fm), effective photochemical efficiency (Fq&#8242;/Fm&#8242;), and non-photochemical quenching (NPQNSV) (Table 1). In general, Fv/Fm and Fq&#8242;/Fm&#8242; are defined as PSII activity8, while NPQNSV&#160;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&#160;by simultaneously measuring recovery kinetics of Fv/Fm, Fq&#8242;/Fm&#8242;, NPQNSV, and &#963;PSII10. 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. M&#252;ller 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 &#181;m), 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acrodisc syringe filter</td>
			<td>Pall Corporation, Ann Arbor, MI, USA</td>
			<td></td>
			<td>0.2 &#956;m pore size</td>
		</tr>
		<tr>
			<td>Act2Run</td>
			<td>CTG Ltd., West Molesey, UK</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin</td>
			<td>Wako</td>
			<td>023-08711</td>
			<td>AF-6 medium</td>
		</tr>
		<tr>
			<td>CaCl2&#183;2H2O</td>
			<td>Wako</td>
			<td>031-25031</td>
			<td>AF-6 medium</td>
		</tr>
		<tr>
			<td>CaCO3</td>
			<td>Wako</td>
			<td>036-00382</td>
			<td>AF-6 medium</td>
		</tr>
		<tr>
			<td>Citric acid</td>
			<td>Wako</td>
			<td>036-05522</td>
			<td>AF-6 medium</td>
		</tr>
		<tr>
			<td>CoCl2&#183;6H2O</td>
			<td>Wako</td>
			<td>036-03682</td>
			<td>AF-6 medium</td>
		</tr>
		<tr>
			<td>Concentrated Chlorella</td>
			<td>Recenttec, Tokyo, Japan</td>
			<td></td>
			<td>20 mg C&#183;mL&#8722;1 ; store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>FastOcean Act2</td>
			<td>CTG Ltd., West Molesey, UK</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fe-citrate</td>
			<td>Wako</td>
			<td>093-00952</td>
			<td>AF-6 medium</td>
		</tr>
		<tr>
			<td>FeCl3&#183;6H2O</td>
			<td>Wako</td>
			<td>091-00872</td>
			<td>AF-6 medium</td>
		</tr>
		<tr>
			<td>HCLP-880PF</td>
			<td>Nippon Medical and Chemical Instruments 
			&#160;Co., Ltd., Osaka, Japan</td>
			<td></td>
			<td>With LED light bulbs</td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>Wako</td>
			<td>160-04292</td>
			<td>AF-6 medium</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Wako</td>
			<td>167-04241</td>
			<td>AF-6 medium</td>
		</tr>
		<tr>
			<td>MgSO4&#183;7H2O</td>
			<td>Wako</td>
			<td>137-00402</td>
			<td>AF-6 medium</td>
		</tr>
		<tr>
			<td>MnCl3&#183;4H2O</td>
			<td>Wako</td>
			<td>139-00722</td>
			<td>AF-6 medium</td>
		</tr>
		<tr>
			<td>Na2EDTA</td>
			<td>Wako</td>
			<td>343-01861</td>
			<td>AF-6 medium</td>
		</tr>
		<tr>
			<td>Na2MoO4</td>
			<td>Wako</td>
			<td>196-02472</td>
			<td>AF-6 medium</td>
		</tr>
		<tr>
			<td>NaNO3</td>
			<td>Wako</td>
			<td>191-02542</td>
			<td>AF-6 medium</td>
		</tr>
		<tr>
			<td>NH4NO3</td>
			<td>Wako</td>
			<td>015-03231</td>
			<td>AF-6 medium</td>
		</tr>
		<tr>
			<td>Plankton Counter</td>
			<td>Matsunami Glass, Osaka, Japan</td>
			<td>S6300</td>
			<td></td>
		</tr>
		<tr>
			<td>Pylex test tube</td>
			<td>CTG Ltd., West Molesey, UK</td>
			<td></td>
			<td>With rim, 16 x 100 mm</td>
		</tr>
		<tr>
			<td>Vit. B1</td>
			<td>Wako</td>
			<td>203-00851</td>
			<td>AF-6 medium</td>
		</tr>
		<tr>
			<td>Vit. B12</td>
			<td>Wako</td>
			<td>226-00343</td>
			<td>AF-6 medium</td>
		</tr>
		<tr>
			<td>Vit. B6</td>
			<td>Wako</td>
			<td>165-05401</td>
			<td>AF-6 medium</td>
		</tr>
		<tr>
			<td>ZnSO4&#183;7H2O</td>
			<td>Wako</td>
			<td>264-00402</td>
			<td>AF-6 medium</td>
		</tr>
	</tbody>
</table>

measuring photophysiology of attached stage of colacium sp. by a cuvette-type fast repetition rate fluorometer

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 (&#963;PSII), maximum photochemical efficiency (Fv/Fm), effective photochemical efficiency (Fq&#8242;/Fm&#8242;), 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.&#160;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. M&#252;ller 1776, Cladocera, Daphniidae) on baseline fluorescence and &#963;PSII, Fv/Fm, Fq&#8242;/Fm&#8242;, 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.

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&#8722;1. However, Fv/Fm and NPQNSV were significantly affected when S. mucronata was 7.5 inds&#183;mL&#8722;1. Therefore, for measuring the photophysiology of Colacium sp. during the attached stage, w...

Watch this Scientific Journal Video about Measuring Photophysiology of Attached Stage of Colacium sp. by a Cuvette-Type Fast Repetition Rate Fluorometer at JoVE.com

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

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

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

Fast repetition rate fluorometer (FRRf) is a beneficial method for measuring photosystem II (PSII) photophysiology and primary productivity. Although FRRf ...

This protocol measures the photosynthetic activity of attached algae, while they are still attaching to the plankton, just as in nature. The main advantage of this technique is that the measurement of photophysiology can be done in real-time without sample distraction. To examine the effects of zooplankton individuals on baseline fluorescence, prepare adult scapholeberis mucronata from the culture without any attached organisms.Now starve the individuals in FLW at 20 degrees Celsius for at least 90 minutes to avoid fluorescence from the gut content. Pour 1.5-milliliters of FWL into a cuvette. Pick up the desired number of s.mucronata individuals using a pipette under an optical microscope at 100-times magnification and transfer them into the cuvette. Add FLW to bring the sample volume up to 2-milliliters and allow it to acclimate under low light at 20 degrees Celsius is for 15 minutes before the fast repetition rate fluorometer measurement. Then click on act to run"to start the measurement and repeat the measurements more than three times per sample.Read the F0 value from the resulting plot. Pick up colacium species with a molted carapace using a pipette under an optical microscope at 100-times magnification and wash them with FLW. Aseptically inoculate colacium species in the AF-6 medium in a 10-milliliter glass tube on a clean bench.Maintain the culture at a NC2 temperature in a growth chamber, shaking the glass tube gently by hand at least once per day to prevent cell settlement. To examine the effects of zooplankton individuals on chlorophyll a fluorescence from colacium species, use adult s. mucronata without any attached organisms.To avoid fluorescence from the gut contents, starve the individuals in FLW for at least 90 minutes. Set up a cuvette-type fast repetition rate fluorometer. Now pour a 1.5-milliliters of sub sample of pre-cultured colacium species into a cuvette.Then transfer the desired number of s. mucronata individuals into these cuvettes and bring the sample volume up to 2-milliliters with the filtered medium. Acclimate the individuals under low light at 20 degrees Celsius for 15 minutes before taking the fast repetition rate fluorometer measurement.Click on act to run"to start the measurement, repeating the measurements more than three times per sample. Read the F0 and FM values from the resulting plot. To correct the baseline fluorescence, filter the culture medium using a 0.2-micrometer pour-sized filter and measure the fluorescence.Subtract F0 of the baseline sample from F0 and FM of colacium species or modify the blank correction value in the settings in the options tab. Isolate s. mucronata individuals with colacium species using a pipette under an optical microscope.Then wash s. mucronata using FLW. Transfer s.mucronata into 100-milliliters of FLW and keep them under dark conditions at NC2 temperature for 90 minutes for starvation. Pour 1.5-milliliters of FLW into a cuvette. Transfer around ten s.mucronata individuals with colacium species into a cuvette and add FLW to bring the sample up to 2-milliliters. Acclimate the individuals under low light at N2C temperature for 15 minutes and measure chlorophyll a fluorescence, as mentioned in the text manuscript. To enumerate the number of attached cells, fix the sample with 2%glutaraldehyde after taking the fast repetition rate fluorometer measurement.Then take pictures at several focal depths and positions of s. mucronata under a light microscope. There was no significant effect on baseline fluorescence or chlorophyll a fluorescence by s.mucronata up to 5 individuals per milliliter. However, maximum photochemical efficiency and non-photochemical quenching were significantly affected when s. mucronata density was 7.5 individuals per milliliter.Seasonal variation in the phytophysiology of colacium species during the attachment stage and the planktonic stage for the stationary phase in the AF-6 medium showed similar mean maximum photochemical efficiency and non-photochemical quenching. For the attached stage of colacium species under dark conditions, there was no significant difference in photophysiological parameters, except non-photochemical quenching being higher for manganese than calcium after three hours, and at a low light intensity at 21 hours. For the planktonic stage, PS2 absorption cross-section was significantly lower in the manganese than calcium treatment at three hours.Effective photochemical efficiency was significantly higher, but non-photochemical quenching was lower in the manganese treatment than control at 21 hours. Under increasing light, manganese tended to decrease PS2 absorption cross-section at three hours, in non-photochemical quenching at 21 hours, while increasing the effective photochemical efficiency at three hours. Calcium slightly improved non-photochemical quenching under increasing light.However, it decreased the effective photochemical efficiency at three hours. The most important thing is the effect of substrate organisms. If algae attached to different species or materials, the effect can be different.In addition to this protocol, measurement of carbon fixation or oxygen production rates are useful to clarify the response of the calcium productivity to the experimental manipulations. This protocol enable us to clarify how the photosynthetic activity of attached algae responds to environmental changes.

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

Abstract