Ciliate Biodiversity and Methods to Identify Ciliates (11 January 2018)

Purpose

The objective of this lab was to give us an outline of what to expect for the course and to introduce us to several methods that can help us identify ciliates based on their genetic information. We also spent the latter part of the class reading articles assigned to our respective groups. We were also tasked with coming up with a 5-7 min presentation about the article.

Procedure

The first part of the class involved some discussion about the importance of studying ciliate biodiversity and the challenges associated with identifying ciliates based on their morphological features. Then, we were provided with Bermuda Grass Plant soil samples which we used to observe ciliates.

1. Using a micropipette, transfer 10µl of the soil sample onto a concavity slide.

2. Observe the sample under a compound microscope.

3. Record the distinct features and characteristics of each ciliate observed.

We were then introduced to three different methods that can allow us to identify ciliates based on their genetic makeup. The three methods are single isolate identification, Metabarcoding, and Metagenomics. My group then read article 5 titled ‘CBOL Protist Working Group: Barcoding Eukaryotic Richness beyond the Animal, Plant, and Fungal Kingdoms’. We then proceeded to answer the questions on our Questions that Matters.

Results

I managed to observe 2 different species of ciliates from the Bermuda Grass sample under the compound microscope. The unique characteristics of the two ciliates are as follow:

Ciliate #1:

• Translucent
• Shaped like a rice grain
• Darts around soil at moderate speed
• Relatively large in size
• Grey specks found on body

Ciliate #2:

• Spherical in shape
• Small in size
• Darts around soil extremely quickly

After reading our article titled ‘CBOL Protist Working Group: Barcoding Eukaryotic Richness beyond the Animal, Plant, and Fungal Kingdoms’, my group and I picked up several very useful information about metabarcoding.

The article is a review article that aims to discuss the advantages and limitations of DNA barcodes currently in use and introduce a two-steps barcoding approach to assess protistan biodiversity.

Challenges of Protist Metabarcoding:

• Multitude of genotypes that are hard to separate
• 18s rDNA is not effective enough to distinguish between interspecies relationships
• No single set of molecular markers has been identified that can be used to identify all species of protists (including ciliates).
• “The large majority of protists are currently uncultivable by known means or unavailable in culture collections”
• Number of catalogued protist species is very low as compared to the total estimated number of protist species.

Useful information that can potentially be applied to our research question:

• 18s rDNA is found in all Eukaryotes
  • many copies
  • highly expressed
  • includes a combination of highly conserved and variable nucleotide sequences
• D1-D2 and/or D2-D3 regions at 5’ end of 28S rDNA positively tested in ciliates
• A two-step method of metabarcoding:
  STEP 1: Preliminary Identification using eukaryotic barcode
  STEP 2: Group-specific barcode for ciliates
• The proWG website has information on protocols and recommendations about protist barcoding.
• The Barcode of Life Database has information about protist multi-locus barcodes.

Conclusion

I am very eager and excited for what is to be expected for this course. It seems to be quite different from what we did the previous semester. I am confident though, that I will learn plenty of new things this semester.