Duckweed experimental procedure and maintenance

Duckweed Experimental Procedure

Counting duckweed individuals

Living individuals: Each duckweed frond to be counted as a single individual frond may be attached to a colony or separated. One frond is any visible protuberance germinating from its parent. In this picture, for example, we have 9 fronds, each labeled with a letter.

Picture source: Laird, R. A., & Barks, P. M. (2018). Skimming the surface: duckweed as a model system in ecology and evolution. American journal of botany, 105(12), 1962-1966. https://bsapubs.onlinelibrary.wiley.com/doi/full/10.1002/ajb2.1194

Dead individuals: fronds are considered as dead when more than ~90% of their surface area has lost pigmentation.

Living and dead fronds should be counted separately, as per the example below:
LM living: 3+5+2
LM dead: 0+1+2

Duckweed generation homogenization (Pre-experimental Procedure)

1. About 14 days before experiments, aseptically transfer about 100 daughter fronds of about the same size and which have not yet reproduced to a graduated 200 mL beaker containing 100 mL of Hoagland's solution. A mother frond and a daughter frond can be distinguished by the size of the root, as mother fronds have longer roots according to our observations. Label the beaker as "generation zero" (as we do not know the order of birth for the daughters we have separated)
2. After reaching maturity (about 6-7 days later), manually separate the first daughter fronds from mother fronds by gently pressing a loop in between fronds, and subsequently transferring them to a new beaker containing growth media. Label the beaker as "generation 1"
3. On the experiment day, select the first daughter from generation 1 mother fronds, so that the plants used in the experiments are considered as "generation 2" fronds. Ensure the first daughter selected for experiments is healthy (bright green) and mature (regular adult sized-frond)

Experimental Setup

Duckweed is to be transferred in the laminar flow room (use lab key to open the door). Gloves are required to avoid contamination.

1. For the flow hood, turn on the light (red power button) and the fan (black turbine) to start the laminar flow

2. Use ethanol to wipe the counter in the laminar flow room with a Kimwipe

3. Dispense ~0.5 mL of the growth medium Hoagland's solution (transparent bottle in large fridge) with a micropipette into each well of a 24-well plate. If using a well plate which already contains nutritional media, complete wells with media as needed. If using a used but empty well plate, spray it with ethanol in the laminar flow room and wait 5 minutes. Next, wipe it using a Kimwipe and proceed with nutritional media filling.

4. Select the newest source culture for field Lemna minor and Landoltia punctata in petri dishes in the duckweed storage incubator

5. Using a sterilized tweezer (stored in the oven), separate a single mature duckweed daughter frond and place it in a well. Daughter fronds can be differentiated from their mothers because they will be attached to either the right or the left side of the meristematic pocket of a mother frond. Their roots will also be smaller. Repeat this process up to a total of 12 fronds of each species. Ensure there are no visible new fronds germinating from the fronds you selected for the experiment

6. Repeat step #5 for each of the 4 well plates which will integrate experiments

7. Place Parafilm over the well plates before placing their lids, and label the well plates with the incubator number and species acronym (LP and field_LM) in a randomized manner

8. Place each well plate in its respective incubator

9. Configure the incubators according to the instructions close to the computer containing Celsius software

10. Write the relevant information on the lab notebook, using the previous week's experiment notes as a template

Experiment End Procedure

1. After 120h, stop incubators

When the red line on the temperature sequence graph reaches the 120h mark (indicated by a blue vertical line), the experiment should be ended, when incubators will be automatically reset it to constant 20C. For each incubator:

1. Click the stop icon (i.e., the red square)
2. Go to file > load tempering profile, then select the corresponding profile name (e.g., Simplelong15_000_10)
3. Go to file > load protocol data, then select the corresponding protocol file name (e.g., e73inc4_15_000_10)
4. Go to file > save protocol data > To report - this should open a window, click to file, this will generate an excel file of the experiment data; name it using the protocol file name, and save Repeat for all incubators

Updating the dataset

The dataset contains the experimental results of aphids and duckweeds, but also the statistics related to the temperature sequence run in the incubators. This includes:

• Categorization: This is determined as positive or negative (for 0.95 autocorrelation only). Essentially it is the trend or slope of the temperature sequence. If the sequence goes from colder to hotter temperatures, it is positive, and vice versa is negative. This categorization is based on the entire temperature sequence.

• Observed mean vs. Set mean: Every temperature sequence has a mean temperature set when the sequence is generated. The observed mean temperature is the mean calculated from the temperatures the incubator actually was at for the duration of the sequence.

• Observed stdev vs. Set stdev: Every temperature sequence has a set standard deviation. The observed standard deviation is the standard deviation calculated from the temperatures the incubator actually was at for the duration of the sequence

• Observed autocorrelation vs set autocorrelation: Every temperature sequence has a set autocorrelation (either 0 or 0.95), the closer it is to 1, the higher the degree of autocorrelation. The observed autocorrelation is calculated from the temperatures the incubator actually was at for the duration of the sequence.

*Gap and Gap length: There have been instances in the past where the incubator would stop recording and outputting the temperature sequence. A gap qualifies as consecutive zeroes for 5h straight or more.

Procedure

• Create a new folder and name it something like "current_experiments"
• Add all the excel files generated from the given experiment to the folder
• Copy the file path for the folder just created
• Open cat_stats.R file in RStudio
• Paste the file path in line 4, in the setwd function
• Run code. This generates an excel file called cat_stats_PorN.csv that will contain all the elements discussed above for each file given (i.e., set vs obs mean, stdev, autocorrelation, cat, etc). The file will be located in the folder made by you
• Open the cat_stats_PorN file and copy all the values from columns starting at Program_mean to Obs_ac, and paste in the dataset
• Copy the categorizations and paste in the cat_1 column in the dataset
• Close cat_stats_PorN file
• Open the cat_stats_PorN, the categorization now should be corresponding to the entire sequence, copy and paste in the cat_1 column in the dataset Note: Files with zero autocorrelation should have N/A as the categorization.

2. In the laminar flow room, perform duckweed counts and note them on the lab notebook

3. Discard the duckweeds (place them in the refrigerator for posterior drying)

4. Erase the labelling and place the well plates back in the storage incubator

** In case of contamination, proceed with counting as usual and note on the notebook (eg., "LP had 2 contaminated wells"). Then, discard the media in the sink, wipe the well plate using a paper towel and discard it in the recyclable garbage. If a new well plate is needed for next experiment, take one from the white box on the floor, close to the cabinet in front of the incubators.

Duckweed photo capture

Procedure to take photos at the beginning and at the end of the experiment:

• Take well plates to the laminar flow room and turn on the fan, but not the laminar flow lights

• Sterilize the laminar flow surface area

• Take 1 photo for each well plate

• Keep the lights of the room on

• Place a white sheet of paper under the plate (there is one close to the laminar flow room computer)

• Open the well plate and place its labeled lid above the plate containing the fronds

• Place a ruler with centimeter measurements up on the right side of the plate, vertically (there is one ruler close to the laminar flow room computer)

• Using a sterilized spatula, move fronds to the middle of the media

• Take a photo from above without using the flash, while trying not to move a lot; the cell phone should be in a horizontal position (please do not incline it to any direction)

• Check if all fronds are appearing in the photo, as sometimes fronds are partially hidden when they are too close to the walls of the wells; re-take photo if needed

• Ensure that all plant contours are clear by zooming in; retake if needed

• Rename each photo as the incubator number (eg., inc2, inc3, etc.)

• Create a new folder corresponding the experiment number (eg., e115, e116, etc.) here: https://drive.google.com/drive/folders/1JTaIjlYubo7RZsXuzb6pdfUVL1ZB2vBW

• Create 2 new subfolders named "initial" and "final" (corresponding to the beginning and the end of the experiment, respectively)

• Upload the photos to the corresponding folder

Duckweed photo analysis

By: Amihan Bularan and Debora Andrade_Pereira

1. Open ImageJ software

2. Drag and drop a given duckweed picture

3. Select the "straight" line tool

4. Select a point on the ruler and drag to another point (e.g., 10cm mark to 11cm mark)

5. Go to Analyze > Set scale; In the pop up window set "Known Distance" to the distance you selected with straight tool (e.g., 1 cm) ; set "Unit of Length" to units you are using (e.g., cm), click ok

6. Go to Image > Adjust > Color Threshold... to bring up this selection window:

7. Use the sliders to get it so only the fronds are in red. A little bit of noise is fine, as we can manually correct it in a later step

8. Press the Select button on the color threshold window to confirm the selection, which highlights everything we selected in yellow

9. We can then zoom in and use the Selection Brush tool to manually change what is highlighted. Most of the time we don't really need to, but sometimes we need to un-select roots or reflections, or fill in holes

10. Click Analyze > Measure

11. Take a screen shot of the manipulated image next to the measured area results

Drying duckweeds

1. After counting fronds and/or taking pictures of well plates, get 12.5 diameter filter papers (2 per well plate, 1 for each species) and fold them in half

2. Using a regular pen, write on top of each filter paper the experiment number, incubator number, and species

3. Place all fronds of a given treatment / species inside the folded filter paper

4. Place the filter papers inside glass petri dishes

5. Dry in the oven at 60°C for 12h

6. The next day, remove dried fronds from the oven and place clothespins around the filter papers to secure dried plants in place

7. Place dried fronds in Ziploc bags, and place these bags in the refrigerator for posterior weighting

Creating a new stock population

Once every month we need to create a new stock population. This is to ensure we have a continuous supply of healthy duckweed.

1. Setup the laminar flow room as outlined in the experimental setup procedure.

2. Throw out oldest stock population. In case it is a non-native species, place it in the petri dish located in the refrigerator for posterior oven drying. Rinse the utilized beaker and set aside for autoclaving.

3. Using a new autoclaved beaker, add 100ml of hoglands solution.

4. Sterilizing frequently, add 30 fronds from the newest stock to the new container.

5. Once completed, cover with 2 layers of parafilm.

6. Label the beaker with the species name and the date.

Discarding non-native specimens

Non-native duckweeds to Canada such as Wolffia globosa or Landoltia punctata are not directly discarded in the sink or garbage. Use kim wipes or a thin paper towel to filter duckweeds from your media, or get them using tweezers or a spatula if in small quantities. Place the wipes or paper towel in a large glass petri dish located in the refrigerator. When this petri dish is full, place the fronds in the oven and dry for 12h at 60°C. The next day, discard the dried content in the garbage and place the petri dish back in the refrigerator.  

Duckweed sterilization procedure

Procedure to transfer field-collected duckweeds to lab conditions (sterilization)

1. Wash duckweed fronds with deionized (DI) water

2. Place in 0.5% v.v. sodium hypochlorite (bleach) solution for a period of time between 30 seconds and 5 minutes (OECD/OCDE guidelines; EPS Canada, 2007)

Household bleach = 5 to 6 % sodium hypochlorite (confirm the expired date of the product we used is within 6 months) (https://www.uwo.ca/animal-research/doc/bleach-sop.pdf)

0.5% v.v. sodium hypochlorite solution: Add 900 mL of water Add 100 mL of household bleach Ensure the contents are mixed thoroughly (https://www.northeastern.edu/ehs/wp-content/uploads/2014/12/Bleach-Fact-Sheet-Draft.ejc2_.pdf)

3. Rinse again in DI water for several times: properly sterilized plants will have a small green area in the bud zone along the center of the frond; estimated rate of survival is 10% (EPS Canada, 2007)

4. Aseptic transfer (creation of new source cultures): in the laminar flow room, aseptically transfer individual duckweed fronds to sterilized test tubes or small vessels filled with a small amount of Hoagland solution (eg., 10mL) (Jewell, Moorsel, & Bell, 2022; https://www.authorea.com/doi/full/10.22541/au.165729067.70397742/v1)

5. Enclose vessels with parafilm and label with species name and sterilization date; place in incubator (20C)

6. Once a week, discard contaminated samples (eg., solution is green or whitish)

7. Before starting experiment: acclimation time options: 28 days (Armitage & Jones, 2019); 7 to 10 days (EPS Canada, 2007)

Alternative procedure to maintain field-collected non-axenic cultures of duckweeds

This procedure is to be applied to the remaining collected fronds that will not be sterilized. Maintain duckweeds in black containers to allow for light penetration only through the top (to avoid further algae proliferation). Change the pond water twice weekly (https://www.jstor.org/stable/43236327?seq=2#metadata_info_tab_contents). Keep duckweed containers under the light (growing cart).