TITLE: Protocol for bacterial depletion of Aiptasia anemones - Towards the generation of gnotobiotic/germ-free cnidarian host animals
AUTHORS: Rúben M Costa, Anny Cárdenas, Christian Voolstra

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol aims to obtain bacteria-depleted Aiptasia polyps. It is divided into 2 sections: (1) a rearing protocol composed of sterile food preparation and anemone priming and (2) a bacteria-depletion protocol for Aiptasia. </div></div>

[STEPS]
?. [Rearing Protocol]
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.
?. [Rearing Protocol]
Prepare the antibiotic solution (ABS) by mixing antibiotics with ASW and sterile filter the final solution through 0.22 µm pore size filters. Cover the bottle with aluminum foil to protect from light.
?. [Preparation of decapsulated Artemia cysts]
To prepare the decapsulated Artemia cysts:
?. [Preparation of decapsulated Artemia cysts]
Weight 2g of cysts and mix with 150 ml of tap water. Let hydrate with aeration for 1h;
?. [Preparation of decapsulated Artemia cysts]
Prepare a saturated brine solution by mixing at least 35% w/v (better >40%) of NaCl in MilliQ water and autoclave it (80 ml). The solution must have a substantial amount of precipitated salts to account for the water that will come out of the hydrated cysts;
?. [Preparation of decapsulated Artemia cysts]
After 1h of hydration, add 150 ml of bleach to the cysts and mix with aeration between 4-6 minutes.
The mixture will become foamy (don’t aerate vigorously!) and the cysts will change color, first from brown to white, then orange. It is more important to watch the color change than to follow an exact time, so the cysts do not become inviable due to overtime in bleach. Still, as a first approximation, don’t leave it longer than 5-6 min.
?. [Preparation of decapsulated Artemia cysts]
When the majority of the cysts look orange, strain them in a 63 µm strainer and rinse the cysts extensively under tap water for at least 1 min; or until they don’t smell like bleach.
?. [Preparation of decapsulated Artemia cysts]
Rinse the cysts for an extra 30 sec in MilliQ water.
?. [Preparation of decapsulated Artemia cysts]
Pour the cysts in a 50 ml polypropylene tube and, in a biosafety cabinet, add the antibiotic solution (ABS) until covered. Incubate in a tilting lab mixer (or belly dancer) for 30 min.
?. [Preparation of decapsulated Artemia cysts]
In the biosafety cabinet, strain the cysts and scoop them to the cool autoclaved saturated brine solution and place the cysts in the fridge. This will de-hydrate them and prevent the hatching process.
?. [Preparation of decapsulated Artemia cysts]
Cysts should last up to 4 months at 4°C.
If salt is not precipitated in the brine solution, add more as the cysts will only preserve while they are de-hydrated.
?. [Priming of Aiptasia animals targeted for bacterial depletion]
Make sure the animals release mucus. This step depletes the bacteria on the surface mucus layer and also washes away parasitic eukaryotes (Figure 3).
?. [Preparation of decapsulated Artemia cysts]
To hatch the cysts, in a biosafety cabinet, shake the brine to place the cysts in suspension, let the salts settle for a couple of seconds, take 1 ml of the suspension and mix with 35 ml of ASW in a 50 ml falcon tube (or a bigger amount onto a sterile hatching system) and incubate at RT with agitation. Cysts will hatch in 24-48h.
In the first hatching, and before feeding Aiptasia animals, take 200 µl of the hatching water after 48 h and plate it in Marine Agar or other rich marine media and incubate for 5 days @ 25°C. Absence of colonies indicates successful treatment. In case of bacterial growth, hatch the cysts in ABS instead of ASW.
The amount of decapsulated cysts can be scaled up using the same protocol in a higher proportion of cysts and solutions, but up to a limit (see (Sorgeloos et al., 1977)).
?. [Priming of Aiptasia animals targeted for bacterial depletion]
Always open the stock of decapsulated cysts under sterile conditions.
?. [Aiptasia bacterial depletion protocol]
Repeat step 4 from the Priming section;
?. [Aiptasia bacterial depletion protocol]
After the polyps released mucus, transfer the polyps to a petri dish with the antibiotic solution (ABS).
All steps after step 7 must be done in a biosafety cabinet, in a sterile environment applying proper sterility practices.
Each antibiotic targets a different mechanism of bacterial replication to ensure different killing mechanisms. The antibiotic solution can be kept at 4°C protected from light during the course of the treatment (1 week).
?. [Aiptasia bacterial depletion protocol]
Wash the Aiptasia animals once more in ABS and leave them in the plate for ~15 min.
?. [Aiptasia bacterial depletion protocol]
Transfer each Aiptasia polyp to a 24-well plate, 1 polyp per well and fill with 1 ml of ABS.
?. [Aiptasia bacterial depletion protocol]
Let the polyps adhere to the wells for a few hours and check in the inverted microscope for the presence of parasites (Figure 3). In case you still see something, wash the well again with ABS.
?. [Aiptasia bacterial depletion protocol]
Treat the polyps for 7 days, with daily media exchange, and incubate @ 25°C under normal light cycle conditions.
?. [Aiptasia bacterial depletion protocol]
In between exchanges, clean the bottom of the wells with sterile cotton tip swabs lightly wet with 70% Ethanol in MilliQ water and rinse 1 time with ASW.
Treatment should be initiated and ASW exchanged at the beginning of the incubator’s dark cycle to prevent early photodegradation of light-sensitive antibiotics (e.g., Rifampicin).
If incubating plates in incubators with high ventilation, put the plates inside a zip lock bag with a wet piece of paper inside to prevent evaporation.
?. [Aiptasia bacterial depletion protocol]
?. [References]
Baumgarten, S., Simakov, O., Esherick, L. Y., Liew, Y. J., Lehnert, E. M., Michell, C. T., et al. (2015). The genome of Aiptasia , a sea anemone model for coral symbiosis. Proc. Natl. Acad. Sci. 112, 201513318. doi:10.1073/pnas.1513318112.Galkiewicz, 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. doi:10.1128/AEM.01303-08.Sorgeloos, P., Bossuyt, E., Laviña, E., Baeza-Mesa, M., and Persoone, G. (1977). Decapsulation of Artemia cysts: A simple technique for the improvement of the use of brine shrimp in aquaculture. Aquaculture 12, 311–315. doi:10.1016/0044-8486(77)90209-5.
?. [Aiptasia bacterial depletion protocol]
To check the efficiency of the treatment, rinse and lysate individual polyps after recovery in ASW using a pestle and plate 50-100 µl of the lysates in Marine Agar plates or another rich marine medium, as well as a drop of water of each well. Absence of bacterial growth after 5 days at 25°C indicates successful treatment.
Thraustochytrids can also grow in the agar plates when animal lysates are plated, although, colonies will only show up after 2/3 weeks of incubation.
?. [Aiptasia bacterial depletion protocol]
Use the remaining lysate to perform DNA extraction and to perform a PCR of the 16S rRNA gene for 30 cycles using 10 ng of DNA template. Absence of amplification or amplification comparable to the PCR negative control in gel electrophoresis indicates successful treatment.
The PCR thermal profile was as follows: 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.
One may consider doing PCRs using cDNA to circumvent amplification of DNA from dead bacteria. Thraustochytrids mitochondrial DNA can also amplify with 16S universal primers.
?. [Aiptasia bacterial depletion protocol]
After 7 days of treatment, recover the animals in ASW for 1 day, washing and exchanging the ASW twice.
It is possible that you’ll see some round structures, between 6-12 µm in size, that burst and release tiny spore-like cells (~2-3 µm). These are Thraustochytrids, and are currently the only protist we cannot eliminate, although, if polyps are kept clean with daily ASW exchange, they should be minimally present.