Brooklyn Urban Ecology and Environment Program (BUEE)

Ana’s TidyData

Ana’s Duckweed Poster

presented by Ana Remis

https://docs.google.com/presentation/d/1iTN9JtwJ_0A_xmIQUZH02htL6Z4ZiFUne7lWX0R73gk/edit#slide=id.p

Ana Sofia Remis

Research Experience for Undergraduates at Brooklyn College

Muth Lab

Abstract

Duckweeds are a family of diverse and useful aquatic plants that grow on the surface of ponds, lakes, and ditches  all over the world. Duckweed’s rapid-growth rate, small genome, natural diversity, and easy culturing make it an excellent model system for studying the hologenome theory of evolution. The hologenome theory purports that the unit of natural selection is not just a singular organism but the sum of an organism and the constituents of its microbiome. In addition duckweed’s extensive phytoremediation potential,, use as a biofuel, and agricultural use as a feedstock make it a valuable organism for study in an ever-urbanizing world facing renewable energy and food production challenges.

Our experiment will genotype the species of duckweed found in the Prospect Park pond. We will also probe the recruitment patterns of duckweed by analyzing the microbes that become associated with sterile duckweed after exposure to Prospect Park pond water and subaqueous sediment (mud).

Overall Questions:

Microbial Composition

Which microbes make up the microbiome of duckweed? Do these microbes vary between different duckweed habitats and duckweed species?

Hologenome

How do bacteria help or hinder their host duckweed? Is the relationship between them symbiotic/ in what ways in particular?What is the influence of the microbiome on duckweed fitness and speciation? How does the microbiome affect duckweed fitness such as growth rates and toxin takeup? Are differences in duckweed microbiome (if any) reflected in characteristics and qualities of the host populations)

Recruitment

What is the role of plant exudate in forming plant microbiomes?Do plant exudates alone attract a microbiome? How does this microbiome differ from that of a microbiome of a plant present along with the exudate?

Experimental Design

Protocol:

Sample Collection / Preparations

1. We grew and maintained sterile cultures of duckweed from the RDSC. Approximately ten fronds were needed for each well so we made sure to have enough of the samples we’d be using for our experiment.  

1. On the afternoon of Friday June 29th we collected water and mud/subaqueous soil from Prospect Park pond locations 1, 2, and a third location (all which had significant populations of duckweed growing) within 48 hours of the experiment start date/point (July 1st). We used a dredger tool thrown approximately 2-3 feet from the edge of the pond to get sample sediment and water and placed our collections in large tupperware, with one tupperware designated and labeled for each site. We collected enough sediment and water for there to be 2 mL of sediment and 8mL of pond water per well for 28 wells (28 wells x 2 mL of sediment per well = 56 total grams of sediment) (28 wells x 8mL pond water = 224mL). Both the water and the sediment sat for two days in a growth chamber set at 22 degrees Celsius, although temperature varied by several degrees throughout the day/night cycle, reaching highs of 26 degrees Celsius.  

Setting up the wells:  

We set up six 6-well plates described in the table below:

The sterile control replicates contain sterile duckweed samples in 10mL of SHBS solution. The experimental replicates receiving treatment contain 2 mL of sediment and 8mL of pond water uniformly mixed from the three Prospect Park locations.

1. Pool/Combine sediment from the three sample locations. Then filter the sediment through a .85mm sieve. Use a 10mL tube to measure out 2 mL of sediment ,then deposit the sediment into appropriate wells.  

2. Combine pond water from the three sample locations. Filter pond water using a pipette to pass the water through 70 and then 40 micron filters to remove algae and debris. Add 8 mL of this filtered pond h20 to each of the appropriate wells.
3. Allow for some hours to pass so that the sediment settles.
4. Add 10 fronds of the appropriate samples to the designated wells. Mark and number the plates for orientation on the side of the bottom tray, top of the bottom tray, and lid of the tray so that samples can be IDed .
5. Photograph for growth/size/surface area comparison using ImageJ
6. Sterile control wells should have 10mL of SHBS and 10 fronds of the sample duckweeds. The turion controls should not have any duckweed, just 2mL of sediment and 8mL of water.

Incubation and Recruitment  

1. Set the incubator to have Day/Night conditions (16hr light, 8 hr dark)  
2. Check on the growth of the samples every 48 hours. Photograph and analyze growth rates by using imageJ surface area analysis or by simply counting the fronds and comparing the numbers.If overgrowth is occurring –remove 10-20%  of the samples to reduce stress
3. Check on the growth of the Treatment 1 samples at 70 hours by removing a few fronds from each well, gently rinsing each sample in 3mL of .5x SHBS media twice, and aspirating off excess liquid. Water and sediment were collected from plate 1 for microbial analysis later.  
4. Freeze Treatment 1 samples in –20 degree Celsius fridge.  

Accessory Experimentation

Genotyping the microbial community of Prospect Park pond water:

Collect roughly 250 mL of unfiltered pond water, uniformly mixed from each site, and place in a vessel.  

Collect roughly 250 mL of pond water, uniformly mixed from each site, and pass through 70 and then 40 micron filters.

Using a vacuum pump and Whatman membrane filters with absorbent pads and a pore size of .45microns, separately pass both the filtered and unfiltered Prospect Park pond water so that you end with two filter membranes that can be processed for microbial and environmental DNA.  

Perform NanoDrop measurements to determine concentration and quality of sample.

Using the DNeasy PowerWater Kit and protocol, prepare samples to be sent off for sequencing.  

Genotyping sediment samples from Prospect Park ponds:

Perform NanoDrop measurements to determine concentration and quality of sample.

Using the DNeasy PowerWater Kit and protocol, prepare samples to be sent off for sequencing.  

Genotyping the duckweed samples from Prospect Park:

Perform NanoDrop measurements to determine concentration and quality of sample.

Using the DNeasy PowerWater Kit and protocol, prepare samples to be sent off for sequencing.

Microscopy of roots systems of duckweed samples

Microbiome DNA extraction

• 48-72 hours and 10-14 days
• Qiagen Power Soil DNA extraction

DNA quantification and purity

• NanoDrop measurements

Send samples for Illumina NextGen sequencing

Total samples to be sequenced: 30

(First time point: 4 treatments per plate x 3 plates = 12. Second time point: All 6 wells per plate x 3 =18)   

• Prepare DNA samples
• Control DNA samples

Remis.IDP

Remis.citiCompletionReport7226046