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About the project
About the project
The project aims to create a dataset of COI genetic sequences of Rhabdocoel species (flatworms) in the Belgian coast. Sampling (1) took place in several beaches and later processed (2) in Marine Station of Ostend, the laboratory facilities of VLIZ. Microscopic identification (3) was later used to select specimens belonging to Rhabdocoel species (infraorders Eukalyptorhynchia and Schizorhynchia). A final step of PCR amplification (4) for COI mitochondrial gene and posterior electrophoresis were performed.
The supervisor of the project was Pascal Hablützel (Senior Science Officer of the Research Division). During the sampling campain, Mattias Boasser collaboration was complete and crucial.
Vlaams Instituut Voor De Zee
Vlaams Instituut Voor De Zee
Wandelaarkaai 7, 8400 Oostende, Belgium (https://goo.gl/maps/QTTqw2kaoZ7eoovM9)
Marine Research projects (http://www.vliz.be/en/research-topics )
Ocean & Human Health
Life's Roots & Rates
Understanding & Optimizing Observations
Nature, Changes and Solutions
Seascapes Past & Future
Maritime Society & History
Integrated Carbon Observation System (ICOS): Monitoring of greenhouse gases balance
LifeWatch: Biodiversity research
Open-data policy: Data need to be made available as much as possible for scientific research both on a national and on an international level (http://www.vliz.be/en/data-policy)
1. Sampling campaing
1. Sampling campaing
Samples were taken during two weeks in the intertidal zone at low tide (±45 min). Wet sand from up to 5 cm deep was sampled with a shovel or directly from the Falcon tube (50 mL). The samples were treated in situ with ethanol or processed in the lab for further analysis (see "2. From the field to the lab")
The detailed sampling procedures can be found in "Floodline protocol" and "Coastline protocol" (see "Protocols and metadata")
Five beaches:
Five beaches:
De Panne
Westende
Ostend / Bredene
De Haan
Knokke
Two areas in each beach:
Two areas in each beach:
Urban U
Natural N
Three locations in each area according to the amount of debris
Three locations in each area according to the amount of debris
High
Intermediate
Low
Low tide is crucial for high abundance of flatworms
Sampling gallery
Sampling gallery
2. From the beach to the lab
2. From the beach to the lab
Storage
Storage
Samples from the Coastline Sampling were processed in the lab for further analysis. Briefly, the sand was soaked in MgCl2 to deattach the flatworms and later filtered through a 63 micrometer sieve. The retained matter, which included the target organisms, was centrifuged and resuspended in order to concentrate them. Finally, every sample was properly annotated and stored in the freezer.
A detailed description of the processing is available on the "Coastline sampling protocol"
1. Sieve
1. Sieve
Meiofauna is retained through a 63 µm sieve.
2. Centrifuge
2. Centrifuge
1000 rpm for 60 sec. The supernatant is discarded and the pellet resuspended for storage
3. Freezer
3. Freezer
Storage at -20ºC for further analysis
Selection of the specimens
Selection of the specimens
Samples taken in Bredene beach during low tide were analysed to gather a collection of different Rhabdocoel species. These were identified and stored for PCR.
Low-magnification microscope
Low-magnification microscope
The specimen was isolated by using a pippete and put on a glass slide to further observation on the high-augment microscope.
3. Rhabdocoela identification
3. Rhabdocoela identification
Flatworms vs. Not flatworms
Flatworms vs. Not flatworms
Nematoda: They have a cuticula (an external protective "skin") easy to observe on the microscope.
Annelida: They have a pair of antennae.
Flatworms: They are usually less elongated than the others and move differently.
Rhabdocoela vs. Proseriata
Rhabdocoela vs. Proseriata
Proseriata species are flatworms that do not belong to Rhabdocoela. They have a statocyst (see picture) in the anterior part of the body, sometimes visible on the low-augment microscope.
Among Rhabdocoela
Among Rhabdocoela
Particular traits are key for identification:
Copulatory organ
Proboscis
Short and helpful guideline for Rhabdocoela identification
Annelid
Nematode
Proseriata
For a detailed identification guideline, check the "Pictorial description"
Proboscis
It is the organ used to seize the prey and feedProboscis
Copulatory organ
Crucial for identification at species level.Copulatory organ
4. COI gene amplification
4. COI gene amplification
COI is a mitochondrial gene widely used for animal genomic identification. Its sequence is usually conserved enough to be representative at species level. However, flatworms have more variability than average, and few sequences have been reported. In this projects, two primers (RhCo1F and RhCo1R) were tested to amplify COI sequence using PCR.
Polymerase Chain Reaction (PCR)
Polymerase Chain Reaction (PCR)
Primers and DNA Polymerase
Primers and DNA Polymerase
Primers are short DNA sequences that bind to the DNA of the organism in the region of interest. In this case, close to gene COI. This "mark" will allow the DNA Polymerase to start the amplification at the target place.
The DNA Polymerase duplicates the template DNA strand several times, resulting in an exponential growth of the sequence of interest.
Primer function
A thermocycler is used to run the PCR. At each cycle, the number of amplicons (duplicated sequences) is doubled. Therefore, in a 30 cycle PCR the number of copies should be more than 1.000.000.000. Although the yield is never 100%, this technique is widely and commonly used in genomics laboratories.
Thermocycler
Thermocycler
Amplification
Amplification
Electrophoresis
Electrophoresis
This step is used to check the quality of the PCR amplification. Using an agarose gel (1.5%) matrix connected to an electric field, the molecules are separated by size. The DNA has a negative charge and moves towards the positive side. When different DNA strands have been amplified, dissimilar bands are observed along the gel.
Equipment
Equipment
The DNA must be stained with a colorant and observed under UV light.
In theory...
In theory...
The fragments of different samples are sorted according to their size.
Our results
Our results
The primers are expected to run the same distance, but no other band was present.
It didn't work, what should we do next?
It didn't work, what should we do next?
Amplify the amplicons: Sometimes the PCR product has low quantity and it cannot be observed with the electrophoresis. Then, we could try to amplify the already-amplified strains.
Change PCR variables: PCR cycles and processes are sensitive to temperature and time. We could try to improve the protocol by testing other conditions.
Prevent random binding: DNA can bind to other surfaces, such as tubes. We could add a product, such as Tween, that avoids this collateral effect.
To be continued...
To be continued...
Nanopore sequencing: minION (Oxford Nanopore Technologies)
Nanopore sequencing: minION (Oxford Nanopore Technologies)
Extremely handy! (and cheap)
The device is connected to the computer and the sample must be simply introduced.Extremely handy! (and cheap)
How does it work?
If you are interested, check out this 4 minutes video, it is amazing how it works!How does it work?
This project is very representative of genomics marine research, from the sea to the computer. A lot is still to be done, but I was lucky to be part of it!
This project is very representative of genomics marine research, from the sea to the computer. A lot is still to be done, but I was lucky to be part of it!
For further questions, I will be happy to be contacted in pol.sorigue@imbrsea.eu
This project has been possible thanks to Pascal Hablützel leadership and Mattias Boasser collaboration.
This website was created by Pol Sorigué for the Online Symposium 2020 of IMBRSea program.
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