TITLE: Protocol for the generation of axenic/bacteria-depleted Symbiodiniaceae cultures
AUTHORS: Rúben M Costa, Cátia Fidalgo, Anny Cárdenas, Jörg C. Frommlet, Christian Voolstra

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left"><span>This protocol produces bacteria-depleted (“axenic”) Symbiodiniaceae cultures. The depletion level achieved using the following methods is comparable to what was previously described as an “axenic” culture (Bieri et al., 2016; Xiang et al., 2013). The present protocol was applied to 10 Symbiodiniaceae strains and proved successful in generating bacteria-depleted cultures after its application, for an extended period of time (up to 4 weeks), characterized by both culture-dependent and -independent techniques. Tested cultures showed normal growth and photosynthetic efficiency after recovery from the treatment. Variations of the protocol were developed to increase the success of bacterial depletion for different microalgal strains and to make it applicable to algal strains that do not grow well on solid media. Treatments can also be performed in 24-well plates in order to facilitate the measurement of F</span><span style = "vertical-align:sub;">v</span><span>/F</span><span style = "vertical-align:sub;">m</span><span>through the plate bottom using a fiber optic cable, in order to track photophysiology. This protocol was developed in parallel with a recently published method (Costa et al., 2019) to generate bacteria-depleted Aiptasia CC7 polyps, in order to have a toolbox at disposal to disassemble the different compartments of an anthozoan metaorganism for functional testing: the bacterial microbiome, the microalgal symbionts, and the anthozoan host animal. All steps of culture manipulation in this protocol must be done under a sterile environment, using common sterile work practices!</span></div></div></div>

[STEPS]
?. [Culture Maintenance]
Prepare artificial seawater (ASW) by mixing reagents in MilliQ water and adjusting the pH of the final volume to 8.0–8.2. Filter through 0.22 µm pore size filter. Alternatively, autoclave natural seawater and filter through 0.22 µm pore size filter (NSW).
?. [Culture Maintenance]
Prepare F/2 medium by diluting it to 1x in ASW or NSW in a biosafety cabinet and filter again through0.22 µm pore size filter. Store the prepared F/2 medium at 4 °C and let it come to room temperature before adding to cultures.
?. [Culture Maintenance]
Split algal cultures every 2 weeks by transferring 1 part of culture to 9 parts fresh F/2 medium.
?. [Antibiotic Treatment]
Prepare the antibiotic F/2 media solution (F/2 ABS):
?. [Antibiotic Treatment]
Mix 50 µg/ml carbenicillin, 50 µg/ml erythromycin, 50 µg/ml rifampicin, 30 µg/ml nalidixic acid and 25 µg/ml nystatin with 1x F/2 media (in ASW) and sterile filter the final solution through 0.22 µm pore size filters.
Cover the bottle with aluminum foil to protect from light. Alternatively, it is possible to filter the stock solutions of antibiotics and 1x F/2 media separately and freshly prepare the working ABS.
?. [Antibiotic Treatment]
Prepare the antibiotic F/2 media (F/2 ABS2) agar plates:
?. [Antibiotic Treatment]
Add the recommended manufacturer’s agar quantity to a defined volume of 1x F/2 medium.
?. [Antibiotic Treatment]
Dissolve the agar and autoclave the mixture for 20 min at 121 °C.
?. [Antibiotic Treatment]
After autoclaving, in a biosafety cabinet, allow the agar mixture to cool down to ~60 °C before adding 50 µg/ml Streptomycin, 100 µg/ml Ampicillin and 200 µg/ml of Gentamycin and mix.
?. [Antibiotic Treatment]
Pour the agar in the plates and allow to solidify. Close the plates and store in a sealed bag at 4 ºC until use.
?. [Antibiotic Treatment]
Inoculate 10 ml of liquid F/2 medium and incubate in a 12 h light: 12 h dark incubator (35–150 µmol photons m−2s−1 of photosynthetically active radiation) at 25 °C or at the optimal temperature of the Symbiodiniaceae strain.
?. [Antibiotic Treatment]
Count the cells with a Neubauer or Nageotte chamber in the microscope, and once the culture reaches at least 105–106cells/ml, transfer 1 ml of the cell suspension to a 1.5 ml reaction tube.
?. [Antibiotic Treatment]
Spin the cell suspension down at low speed (5 min, 800 x g) and rinse the pellet twice with ASW.
Centrifugation at low speeds decreases the pelleting of bacteria (in comparison to microalgal cells), reducing the bacterial cargo of the initial inoculum.
To check for initial bacterial load and to assess bacterial load throughout the protocol, collect aliquots as described in step 16 and compare all samples during step 17.
?. [Antibiotic Treatment]
Resuspend the pellet in 1 ml of F/2 and inoculate in 9 ml of F/2 ABS.
Each antibiotic targets a different mechanism of bacterial replication. Thus, the cocktail combines different bacteriostatic and bactericidal mechanisms. Nystatin targets fungal contamination. The antibiotic solution can be kept at 4 °C protected from light (but no longer than 2 weeks).
?. [Antibiotic Treatment]
Incubate at the same light and temperature conditions for 7 days.
Check the cultures with an epifluorescence inverted microscope with a filter/cube that detects chlorophyll. Symbiodiniaceae will lose photosynthetic efficiency (loss of red fluorescence), but will survive. Measurements of maximum quantum yield by PAM fluorometry also showed a strong effect of this cocktail on the photophysiology of cells but quantum yield recovered during steps 12, 13.
?. [Antibiotic Treatment]
After 7 days of treatment, collect 1 ml of the cell suspension and repeat step 8.
?. [Antibiotic Treatment]
Resuspend the rinsed cells in 10 ml of F/2 to recover and incubate as before.
?. [Antibiotic Treatment]
Once the culture reaches at least 105–106 cells/ml, take 1 ml and repeat step 8.
?. [Antibiotic Treatment]
Resuspend the pellet in 1 ml of F/2 and spread 50–100 µl in F/2 ABS2 agar plates. Wrap with parafilm and incubate at optimal growth temperature and light cycle until colonies form.
This can take 1-3 weeks depending on the strain.
?. [Antibiotic Treatment]
Pick one colony from the previous plate, resuspend it in 100 µl of F/2 and repeat step 14.
?. [Antibiotic Treatment]
Pick a colony and inoculate 10 ml of F/2 medium. Let it grow until it reaches at least 105–106 cells/ml.
?. [Antibiotic Treatment]
To check for depletion, remove 1 ml of culture and plate 200 µl of the supernatant in Marine Agar or Tryptic Soy Agar. Do the same thing with the original cultures as a positive control.
?. [Antibiotic Treatment]
If the plates show no growth after 5–7 days of incubation (but extended incubation (>14 days) is advised), perform one more split (same way, 1:9) of the Symbiodiniaceae cultures.
?. [Antibiotic Treatment]
Once they reach the desired concentration, collect 1 ml of the suspension, spin it down at high speed (≥ 14 000 x g for 5-15 min), discard the supernatant and perform DNA extraction on the pellet.
For quicker assessment, pellets were resuspended in TE buffer to algal cell concentration of 200.000 cells/ml based on the cell counts. 20 µl of these normalized suspensions were then boiled for 25 min at 100 ºC. The lysate can be used directly as template in step 20. Regular DNA extraction methods can be used instead.
?. [Antibiotic Treatment]
Perform a PCR of the 16S rRNA gene using 10 ng of DNA template. If using preparation methods where absolute DNA quantification is not recommended (See Note in step 19), use algal cell numbers as abasis to normalize across tested conditions. Absence of amplification or amplification comparable to the negative PCR control in an electrophoresis indicates successful treatment.
Alternatively, this profile was used (primer pair 27F-1492R): 94°C for 3 min, followed by 35 cycles of each: 1 min at 94°C, 30 s at 55°C, and 90 s at 72°C. A final extension step was set at 72°C for 10 min.
The PCR thermal profiles used were as follows (primer paris 27F-1492R and 67F-1542R):95°C for 15 min, followed by 30 cycles of each: 30 s at 95°C, 90 s at 55°C, and 90 s at 72°C. A final extension step was set at 72°C for 10 min
?. [Antibiotic Treatment]
ABCDEFG1CultureSpecies
  or ITS2 typeCulture
  collection IDStrain
  OriginPreviously
  reported as “axenic”?ObservationsReference2370Symbiodinium microadriaticumCCMP2467NCMANo-LaJeunesse, 20013-Symbiodinium linucheaeSSA01Pringle LabYes-Bieri et al., 20164-Breviolum minutumSSB01Pringle LabYes-Xiang et al., 2013574Breviolum minutum-Warner LabNo-LaJeunesse, 2001689A2-Warner LabNoStrain does not grow well on solid mediumLaJeunesse, 20017146Breviolum pseudominutum CCMP3450Warner LabNo146 & RT146 are cultures of the same strain; strain
  did not grow well on solid mediumParkinson et al., 20158RT 146Breviolum pseudominutum CCMP3450LaJeunesse LabNo146 & RT146 are cultures of the same strain; strain
  did not grow well on solid mediumParkinson et al., 20159351Breviolum minutum-Warner LabNo-Parkinson et al., 201510MfA2-Warner LabNoStrain did not grow well on solid mediumSteinke et al., 201111Mac702B2-Warner LabNo-Santos et al., 2001
ABCDEFG1CultureSpecies
  or ITS2 typeCulture
  collection IDStrain
  OriginPreviously
  reported as “axenic”?ObservationsReference2370Symbiodinium microadriaticumCCMP2467NCMANo-LaJeunesse, 20013-Symbiodinium linucheaeSSA01Pringle LabYes-Bieri et al., 20164-Breviolum minutumSSB01Pringle LabYes-Xiang et al., 2013574Breviolum minutum-Warner LabNo-LaJeunesse, 2001689A2-Warner LabNoStrain does not grow well on solid mediumLaJeunesse, 20017146Breviolum pseudominutum CCMP3450Warner LabNo146 & RT146 are cultures of the same strain; strain
  did not grow well on solid mediumParkinson et al., 20158RT 146Breviolum pseudominutum CCMP3450LaJeunesse LabNo146 & RT146 are cultures of the same strain; strain
  did not grow well on solid mediumParkinson et al., 20159351Breviolum minutum-Warner LabNo-Parkinson et al., 201510MfA2-Warner LabNoStrain did not grow well on solid mediumSteinke et al., 201111Mac702B2-Warner LabNo-Santos et al., 2001
?. [Variations to the protocol outlined above]
Variation 1 - To increase the success of the protocol by increasing the stringency of the treatment, the first antibiotics treatment (steps 6-10) can be repeated before moving to step 14, without compromising viability of algal cultures. For instance, strains 89 and RT146 still yielded colonies of Stappia indica and Muricauda aquimarina /Paracoccus homiensis, after one round of antibiotics treatment. By contrast, no bacterial growth was detected on marine agar plates (steps 16 to 18) when steps 9-13 were performed again, i.e. twice.
?. [Variations to the protocol outlined above]
Variation 2 - The second antibiotics cocktail (ABS2) can also be applied in liquid F/2 medium, which can be an advantage or might even be necessary when algal strains do not grow well on solid medium. From the tested strains, 89, 146, RT146 and Mf do not grow well on solid medium and depletion of bacteria was only achieved by applying the ABS2 treatment in liquid medium. However, sterility tests after several weeks following the protocol suggest that colony picking from solid medium increases the chance of gaining axenic cultures or at least extends the duration during which cultures can be considered bacteria-depleted. This effect might be due to the physical exclusion of bacterial contamination when a single isolated algal colony is picked multiple times in solid media, versus a suspension culture split, which always carries part of the previous culture.
?. [References]
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							Rúben M Costa, Anny Cárdenas, Christian Voolstra (2019). Protocol for bacterial depletion of Aiptasia anemones - Towards the generation of gnotobiotic/germ-free cnidarian host animals. protocols.iodx.doi.org/10.17504/protocols.io.7mrhk56Galkiewicz, J. P., and Kellogg, C. A. (2008). Cross-kingdom amplification using Bacteria-specific primers: Complications for studies of coral microbial ecology. Appl. Environ. Microbiol. 74, 7828–7831.
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							LaJeunesse, T. C. (2001). Investigating the biodiversity, ecology, and phylogeny of endosymbiotic dinoflagellates in the genus Symbiodinium using the ITS region: in search of a “species” level marker. J. Phycol. 37, 866–880. 
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							Parkinson, J. E., Coffroth, M. A., and LaJeunesse, T. C. (2015). New species of Clade B Symbiodinium (Dinophyceae) from the greater Caribbean belong to different functional guilds: S. aenigmaticum sp. nov. , S. antillogorgium sp. nov., S. endomadracis sp. nov., and S. pseudominutum sp. J. Phycol. 51, 850–858. 
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							Steinke, M., Brading, P., Kerrison, P., Warner, M. E., and Suggett, D. J. (2011). Concentrations of dimethylsulfoniopropionate and dimethyl sulfide are strain-specific in symbiotic dinoflagellates (Symbiodinium sp., Dinophyceae). J. Phycol. 47, 775–783. 
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