Name: measuring photophysiology of attached stage of colacium sp. by a cuvette-type fast repetition rate fluorometer
Uploaded: 2021-11-12T13:00:00
Description: 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

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 ...

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