Jun 15, 2020
Collect of Collodarian (Rhizaria, Radiolaria) nuclei for genomic analyses
- Estelle Bigeard1,
- Loïc Pillet2,
- John Burns2,
- Fabrice Not3
- 1Station Biologique de Roscoff, France;
- 2AD2M, Station Biologique de Roscoff, CNRS, SU;
- 3CNRS & Sorbonne University - Station Biologique de Roscoff
- Ecology of Marine Plankton (ECOMAP) team - Roscoff
- Roscoff Culture Collection
Protocol Citation: Estelle Bigeard, Loïc Pillet, John Burns, Fabrice Not 2020. Collect of Collodarian (Rhizaria, Radiolaria) nuclei for genomic analyses. protocols.io https://dx.doi.org/10.17504/protocols.io.5kgg4tw
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 17, 2019
Last Modified: June 15, 2020
Protocol Integer ID: 25960
Abstract
Collodaria are ubiquitous and abundant marine radiolarian (Rhizaria) protists (Biard et al. 2015). They occur as large colonies (a few millimeters up to 3 meters long) or as solitary specimens. Collodarians are known to play an important role in oceanic food webs both as active predators and as hosts of intracellular endosymbiotic microalgae primarily belonging to the dinoflagellate genus Brandtodinium. Despite their important ecological roles, very little is known about their diversity and evolution. Taxonomic delineation of collodarians is challenging and only a few species have been genetically characterized.
Most Collodaria form colonies comprising tens to hundreds of individual radiolarian cells (i.e. central capsules) embedded in a gelatinous matrix. Each central capsule contains genomic DNA of the Collodaria host while the gelatinous matrix which also contains the DNA of prey and symbionts.
Figure 1: Cells (i.e. central capsules) are distributed in the matrix forming a well-defined compartment. Central capsules, appearing bright under the microscope, measure from 100 to 150 μm in diameter. The dinoflagellate symbionts are enclosed in cytoplasmic structures, either localized within the gelatinous matrix or closely associated to the central capsules. (from Villar et al. 2018)
Figure 2a: 1 part of a Collodaria colony. Each cells is visible thanks to its central capsule (white dots) containing its nucleus. ©Pictures IMPEKAB - E. Bigeard - VFR2016
Figure 2b: Magnified view of a single central capsules with surrounding spicules (colorless) and symbionts (yellow). ©Pictures - F. Not
Some species build a shell-like skeleton aroundtheir central capsule while others have siliceous spicules, similar to those in sponges, in the matrix, and some lack mineral structures altogether. Current taxonomic classification reveals several clades : Sphaerozoidae (skeleton-less but spicule-bearing), Collosphaeridae (mix of skeleton-bearing and skeleton-less taxa), Collophidiidae (skeleton-less). The family Thalassicollidae is composed exclusively of solitary species.
This protocol describes a method for isolating central capsules containing oly the genomic DNA of the collodarian host by removing prey and symbionts through targeted dissolution of the gelatinous matrix and removal all material outside of host central capsules.
Guidelines
protocol EB-BM-MO-016
Villar E, Dani V, Bigeard E, Linhart T, MendezSandin M, Bachy C, Six C, Lombard F, Sabourault C & Not F (2018). Chloroplasts of symbiotic microalgae remain active during bleaching induced by thermal stress in Collodaria(Radiolaria) doi:10.1101/263053
Biard T, Bigeard E, Audic S, Poulain J, Gutierrez-Rodriguez A, Pesant S, Stemmann L & Not F (2017). Biogeography and diversity of Collodaria (Radiolaria) in the global ocean. ISME Journal, doi:10.1038/ismej.2017.12
Biard T, Stemmann L, Picheral M, Mayot N, Vandromme P, Hause H, Gorsky G, Guidi L, Kiko R & Not F (2016). In situ imaging reveals the biomass of giant protists in the global oceans. Nature, doi:10.1038/nature17652
Biard T., Pillet L., Decelle J., Poirier C., Suzuki N. & Not F (2015). Towards an Integrative Morpho-molecular Classification of the Collodaria (Polycystinea, Radiolaria). Protist, doi:10.1016/j.protis.2015.05.002
Lee et al. (2007), Monitoring Repair of DNA Damage in Cell Lines and Human Peripheral Blood Mononuclear Cells, Anal Biochem. doi: 10.1016/j.ab.2007.03.016
Materials
Chemicals:
Sucrose Ref S0389 - Sigma-Aldrich
Spermine Ref S3256 - Sigma-Aldrich
Spermidine Ref 85558 - Sigma-Aldrich
NaCl Ref S9888 - Sigma-Aldrich
KCl Ref P9333 - Sigma-Aldrich
Tris HCl Ref T5941 - Sigma-Aldrich
EDTA 0.5M pH8 Ref 03690 - Sigma-Aldrich
Igepal CA-630 Ref I8896 - Sigma-Aldrich
PBS Ref P3744-12PAK - Sigma-Aldrich
Supplies:
6wells - plate Ref CC7672-7506 - Starlab
40µ sieve Ref 010198 - Dominique Dutscher
Petri dishes Ref 632191 - Dominique Dutscher
Solutions
Solutions
Preparation of Solutions
Sucrose 3M (M = 342.3g / mol)
112.96g in 110ml water milliQ
Store at room temperature
Spermine 0.1M (M = 202.34g / mol)
Powder stored at 4 ° C.
Weigh 40.5mg in a 15ml Falcon.
then add 2ml water milliQ.
Preparation instructions: This product is soluble in water (50 mg / ml), yielding a clear, colorless to light yellow solution.
Storage / Stability: Store at 2-8 ° C.
Solutions of spermine free base are readily oxidized. Solutions are most stable when prepared in degassed water and stored in frozen aliquots, under argon or nitrogen gas.
Spermidine 0.05M (M = 145.25g / mol)
Liquid stored at room temperature.
Weigh 87.15mg in a 15ml Falcon.
then add 12ml water milliQ.
Preparation instructions: Spermidine is soluble in water (50 mg / ml), ethanol, and ether.
Storage / Stability: Spermidine is very hygroscopic and air sensitive. A solution can be formed for storage by dissolving 1.45 g in 10 ml of water and then sterilizing with a 0.22 μm filter.
Store this solution as single-use aliquots at -20 ° C for no longer than one month.
4M NaCl (M = 58.44g / mol)
Weigh 0.47g of NaCl.
Add 2ml of milliQ water.
Store at room temperature.
KCl 5M (M = 74.5513 / mol)
Weigh 2.6g of KCl.
Add 7ml of milliQ water.
Store at room temperature.
Tris HCl 1M pH8 (M = 157.60 / mol)
Weigh 15.76g of Tris HCl.
Make up to 100ml of milliQ water.
Tamp to pH8.
Store at room temperature.
EDTA 0.5M pH8 (M = 292.24 / mol)
Weigh 15g of EDTA.
Add 50ml of milliQ water.
Add NaOH pellets until a pH of 8 is reached.
QSP 100ml of water milliQ.
Filter on 0.2μm.
Store at room temperature.
For 500ml of Lysis Buffer Solution
| Product | Initial concentration | Final concentration | volume | |
| Sucrose | 3M | 0.3M | 50ml | |
| KCl | 5M | 60mM | 6ml | |
| NaCl | 4M | 15mM | 1.875ml | |
| Tris HCl pH8 | 1M | 60mM | 30ml | |
| Spermidine | 0.05M | 0.5mM | 5ml | |
| Spermine | 0.1M | 0.15mM | 750μl | |
| EDTA | 0.5M | 2mM | 2ml | |
| Igepal CA-630 | 2.5ml | |||
| Water milliQ | Qsp 500ml |
Table 1: Recipes for Lysis Buffer Solution
For 500ml of Wash Buffer solution
| Product | Initial concentration | Final concentration | volume | |
| Sucrose | 3M | 0.3M | 50ml | |
| KCl | 5M | 60mM | 6ml | |
| NaCl | 4M | 15mM | 1.875ml | |
| Tris HCl pH8 | 1M | 60mM | 30ml | |
| Spermidine | 0.05M | 0.5mM | 5ml | |
| Spermine | 0.1M | 0.15mM | 750μl | |
| EDTA | 0.5M | 2mM | 2ml | |
| Water milliQ | Qsp 500ml |
Table 2: Recipes for Wash Buffer Solution
Selection of colonies
Selection of colonies
Sort several colonies (maximum 20) according to their morphological type (segmented round shape, segmented straight form, non-segmented, purple color, blue dots, etc.) assuming it corresponds to a single species. Observe using a stereomicroscope and take pictures in wide field and zoom.
Figure 3 : Pictures from 5 different types of colladarians (Types A; B; C; D purple colonies & E colonies with blue dots) - ©Pictures IMPEKAB - E. Bigeard - VFR2016
Lysis of colonies
Lysis of colonies
In a 6-well plate, deposit 8 ml of Lysis solution per well.
Add a 40μm diameter sieve previously rinsed with milliQ water per well. Place about 10 medium sized colonies (1.5 - 2 cm) per sieve. Incubate for 30 minutes at room temperature.
Take a sieve from a well, place it in a 90mm diameter petri dish containing Lysis solution.
Tap the sieve and shake circles with the sieve in the BP.
Repeat several times until the matrix breaks up and is released from the sieve (about 3 minutes).
Figure 4: Plates containing lysis buffer and sieves with colonies and BP containing wash buffer - ©Pictures IMPEKAB - E. Bigeard - VFR2016
For purple colonies and those with blue dots, just one lysis will suffice. For other types, perform with a second lysis as below.
In a 6-well plate, deposit 8 ml of Lysis solution per well. Place the sieve containing the colonies at the beginning of lysis.
Incubate again for 30 minutes at room temperature.
Washing of central capsules
Washing of central capsules
Remove the sieve from the well and place it in the petri dish containing Wash solution.
Tap the sieve and shake circles with the sieve in the BP.
Figure 5 : Washing of collodarian central capsules - ©Pictures IMPEKAB - E. Bigeard - VFR2016
Washing of nuclei present in the sieve.
Repeat several times until the matrix is completely removed.
Observe and control with a binocular loupe (capsule size with respect to the pores, cleanliness of the sample, etc.).
Take pictures If OK.
Rinsing & Concentration of nuclei
Rinsing & Concentration of nuclei
Rinse the capsules in 1x PBS solution or in 0.2µm filtered and autoclaved seawater in a 90mm diameter BP.
Remove the capsules from the sieve by rinsing it with 0.2µm filtered seawater or 1x PBS solution.
Place the capsules in a 1.5ml microtube (or 2ml microtube if necessary).
Centrifuge at 1000 rcf 10 minutes.
Remove the supernatant.
Figure 6: Pellet of central capsules - ©Pictures IMPEKAB - E. Bigeard - VFR2016
Flash freezer.
Storage at -80 ° C.
If using of sea water in the above steps, it is better to rinse with PBS before the DNA extraction to remove salt:
Rinse with 1x PBS solution by depositing 500 μl of PBS solution into the microtube.
Tap gently.
Centrifuge at 1000rccf 10 minutes.
Remove the supernatant.
Repeat once if necessary.
Analyses
Analyses
The DNA extraction method will be published under a separate protocols.io (in collaboration with Genoscope).