Aug 03, 2022
Injection of algae into Ambystomid (salamander) embryo
- John Burns1,
- Hui Yang2,
- Joost S Mansour3
- 1Bigelow Laboratory for Ocean Sciences;
- 2University of Arizona, Molecular and Cellular Biology;
- 3University of Arizona
Protocol Citation: John Burns, Hui Yang, Joost S Mansour 2022. Injection of algae into Ambystomid (salamander) embryo. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl89y17v2w/v1
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 26, 2022
Last Modified: August 03, 2022
Protocol Integer ID: 67676
Keywords: micro injection, alga, symbiosis, salamander, artificial, manipulation
Funders Acknowledgement:
Moore Foundation - Solar Salamanders? Algal Invasion of a Vertebrate
Grant ID: GBMF5604
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Abstract
This protocol describes the steps taken to artificially introduce algal cells into salamander tissues by microinjection for the study of salamander-alga endosymbiosis. Microinjection allows experimental manipulation of the separate partners before the establishment of symbiosis. For instance, algae can be grown with a heavy nitrogen (15N) or carbon (13C) label prior to injections to trace nutrient transfer.
Guidelines
Embryos (A. maculatum) for injection should be between stages 26-31. Injections of younger embryos (~stage 19) ended with the death of all embryos. Injection of embryos at stage 33+ can be problematic because as the embryos flex, they can tear the injection site, preventing the wound from healing and causing eventual death.
Injection of embryos at stages 26-30 has good survival: 95% survival at 24 hours, >50% after 5 days
Materials
- Salamander eggs
- Dense Oophila algal cultures in a growth state (cultures should have mostly motile algae) --> 180,000 cells per mL or more.
- Small steel strainer
- 70% ethanol in a squeeze bottle
- Milli-Q water in a squeeze bottle
- Binocular dissecting microscope
- Tweezers (or needles to control and limit the eggs movements)
- Microinjection apparatus, such as a 3-axis micromanipulator with a nanofil syringe attached (https://www.wpiinc.com/var-3167-sub-microliter-injection-system).
- 35 gauge steel needle (beveled) for nanofil syringe (https://www.wpiinc.com/var-3184-nanofil-needles).
- Optional: motorized injection controller.
- 24 well plate
- Petri dishes
- Plastic transfer pipette, cut such that the opening is large enough to hold one egg.
Before start
Salamander egg clutches need to be collected as close to laying as possible (i.e. very young). Collected egg clutches can be cleaned following the same steps as the first section of this protocol. Once washed the egg clutches should be incubated in the dark to prevent algal growth. This way there should be no algae other than the ones injected during this protocol. Leave the egg clutches to incubate and grow to the desired stage.
Prepare the embryos
Prepare the embryos
Load whole eggs containing embryos into a strainer.
Whole eggs will be washed with 70% ethanol prior to decapsulating the embryos to remove external microorganisms. The ethanol wash would kill embryos, so the eggs must be intact at this point. For A. mexacanum eggs, individual jellied eggs can be used. For A. maculatum eggs, intact eggs need to be removed from the egg mass before being placed in the strainer.
Wash eggs with 70% ethanol for ~20 seconds.
Squirt 70% ethanol over eggs in strainer while rotating strainer around to make sure to thoroughly wash all eggs.
Wash eggs with Milli-Q water for ~20 seconds.
Note
Milli-Q water is used to wash off the ethanol
*wash steps can be repeated if desired.
Following the water rinse, transfer eggs to a 100 mm petri dish containing sterile 20% Holtfreter's solution.
Note
Embryos do not need to be decapsulated and are better of left in the egg.
Embryos are now ready for injection.
Prepare the algae
Prepare the algae
Pour 40 mL of algal culture into a 50 mL falcon tube.
To pellet the algae centrifuge at 300 x g, 00:08:00
8m
Remove supernatant, and place tube upside-down on a kimwipe to remove excess liquid.
Add 20 mL sterile 20% Holtfreter's solution to the algal pellet and resuspend it.
Repeat centrifuge at 300 x g, 00:08:00
8m
Remove supernatant, remove excess liquid as before (go to step #8 )
Note
The centrifugation and wash steps have the effects of:
(a) concentrating the algae
(b) removing the high-nutrient growth medium
(c) removing many contaminating bacteria as they do not pellet well at 300xg.
Resuspend the pellet in 400 uL of 20% Holtfreter's solution.
This is the 100x concentrated algal solution that will be used for injections.
Note
Depending on the algal density this resuspension can be fone into a larger volume
Fix a sample of the concentrated algal culture in 0.1% glutaraldehyde for cell counts. This can be used to verify the concentration of the culture finally used.
Note
Or another preferred fixing method for cell counting
Inject the embryos with algae
Inject the embryos with algae
Prepare an individual embryo in a well in plastic mold (e.g., Fisherbrand™ Disposable Base Molds or any kind of well that will hold the egg snug).
Eggs are typically kept and injected underwater (20% Holtfreter's)
Place the embryo in its mold/well on the dissecting microscope.
Prepare the syringe by backfilling the syringe with the concentrated algal solution. Close the syringe by adding the needle.
Prep the needle by evacuating air, and push out some solution to ensure that algae are coming out during injections.
Carefully penetrate the egg and then break into the embryo's skin surface with the needle (beveled). It is not necessary to push the needle deep into the embryo.
Note
Indeed it is very disadvantageous for the needle to pierce through the other side of the embryo.
Note
Be careful during egg and embryo penetration to NOT hit the needle onto other surfaces and bent or break it.
Inject algae.
Note
For stage 30 embryos, a good quantity of algal concentrate for injection seems to be about 300 nanoliters (0.3 uL).
Carefully withdraw the needle straight back.
Transfer the embryo to an individual well in a 24 well plate containing 1.5 mL 20% Holtfreter's solution.
Repeat until all embryos have been injected (go to step #18 ).
Video of algal injection. Example of algal injection into an early (21-23) stage A. maculatum embryo. Later stages such as 25-28 are recommended.
Place the 24-well plate containing the injected embryos in an incubator with a 12/12 light-dark cycle.
Note
Most embryos should survive and heal after a few (3-5) days.