The Golden Apple Snail Pomacea canaliculata: From Zygotes to Stable Mutant Lines

The scope of this work is to share the methods we developed to generate apple snail stable mutant lines to study gene function. The next experimental challenge is to generate transgenic apple snails through stable DNA insertion in their genome, allowing for linear tracing experiments. To begin, replace daily five to 10%of the water in each tank of a recirculation system housing apple snails.

Siphon the bottom of the tanks to remove debris. Feed the snails three times per week with organic lettuce. Collect the egg clutches from the tanks every day in Petri dishes.

Transfer freshly laid clutches from the tank walls to a dry environment for optimal development. Observe the egg clutches as they transition in color from bright pink to pale gray in about 12 days. Once the clutches are mostly gray, immerse fingers in system water and gently crush them to manually hatch the eggs.

The outer casing will float while the hatchlings sink. For sex determination, place snails aged 1.5 to 2.5 months in ice for 10 minutes to relax their muscles. Using a hemostat locking scissor clamp, pull the operculum anteriorly, ventrally, and posteriorly to expose the head of the snail.

Examine for sex-specific features to determine the sex. The male possesses a small pink penial bulb, a larger pale orange penis pouch, and a large white penis sheath, while the female possesses tubular oviduct and rectum. Return the sexed snails to system water.

If the mantle detaches from the shell during the operation, stop and avoid further stretching. To prepare and test the perivitelline fluid extract, or EPVF, first, clean the workspace and tweezers with 70%ethanol. Transfer clutches aged one to two days post-fertilization into a 50 milliliter tube containing 40 milliliters of 0.8%sodium hypochlorite for three minutes, then rinse the clutches twice with autoclaved distilled water.

Incubate the clutches in fresh autoclave distilled water for two minutes to remove residual sodium hypochlorite. Lay the tube horizontally during the incubations. Remove the water from the 50 milliliter tube and transfer the dry clutches to a Petri dish using forceps.

Using tweezers, crush all the capsules in the clutch. Use curved forceps to transfer the crushed material into two milliliter centrifuge tubes, then centrifuge the tubes at 21, 000 G for 40 minutes at four degrees Celsius. Observe three distinct layers after centrifugation.

Transfer only the top clear pink layer into a fresh 1.5 milliliter tube without combining extracts from different clutches. For collecting wild type embryos, add 40 milliliters of PCEM to a Petri dish containing sterile two day old clutches and use tweezers to mix the broken capsules to dislodge the embryos from the PVF. Under transmitted lights on a stereo microscope, collect the released embryos, then transfer the embryos into a clean Petri dish or four-well plate prefilled with 5%fetal bovine serum in PCEM.

To test the EPVF, place the lid of a 35 millimeter Petri dish facing up inside a 60 millimeter Petri dish. Pipette two 60 microliter drops, each three millimeters in diameter, from each EPVF aliquot into the lid. With a P20 pipette, transfer three to four healthy embryos into each EPVF drop.

Then, pipette three milliliters of paraffin oil to cover the drops, starting between the adjacent drops and then fully covering them. Check daily for embryo development, formation of air bubbles over EPVF drops, and signs of contamination. For microinjection of the zygotes, first, collect freshly laid clutches into a 50 milliliter tube containing freshly prepared L-cysteine solution.

Pour 20 milliliters of the solution and the capsules into a 100 millimeter Petri dish. Then, add 20 milliliters of PCEM to the dish. With a pair of tweezers and a stereo microscope set to 10X, open the capsules one by one, tearing the external membrane.

When the PVF is exposed to the L-cysteine solution, gently swirl the contents of the dish. After letting it settle down for two minutes, use a P20 pipette to collect the released embryos under a stereo microscope. Now, using an inverted microscope equipped with a 20X differential interface contrast objective, micromanipulators, microinjector, pinpoint cell penetrator, and a cold plate, open the needle, breaking its tip against the holding pipette.

Place one of the collected zygote dishes at room temperature and the other at four degrees Celsius to slow development. Add a large drop of 5%fetal bovine serum in PCEM onto depression slides. Then, move 10 to 20 embryos from the room temperature dish to the depression slide.

After placing embryos on the slide, move the leftover embryo dish to four degrees Celsius. Secure one embryo at a time using the holding pipette, aligning the needle and embryo membrane in the same focal plane. Inject the embryos by alternating an injection and a pulse until the needle enters the embryo.

If the needle is too dull, the embryo may die after penetration. If too sharp, the embryo may lyse on contact. Withdraw the needle quickly and firmly without stalling inside the embryo.

Before the end of the day, culture ex ovo the embryos that are both dividing and fluorescent using high quality EPVF. After approximately 11 to 13 days, when embryos are too large for the drops, pipette them out of the culture. After three to five days, feed the hatchlings small pieces of lettuce.

Once F1 juveniles reach at least six millimeters in diameter, wait for tentacle exposure, then cut the tip of one tentacle with micro scissors. Transfer the snail to a six-well plate and the tentacle in 50 microliters of lysis buffer in a PCR tube before sample processing. Formation of air bubbles on top of the EPVF droplets was observed during the first 48 hours of ex ovo culture.

By six days post-fertilization, embryos exhibited normal development display features of early organogenesis, including heartbeat. By nine days post-fertilization, embryos displayed normal development with shell, foot, eyes, and tentacles, and were seen actively moving in the droplet. Contaminants and debris were observed grouped into one mass by the embryos at nine days post-fertilization, and contamination remained under control at this stage.

Partial or complete drying of the EPVF showed to impair proper embryo development, particularly when the paraffin oil overlay was improperly applied or air bubbles were not removed. Contaminated droplets showed darker, irregular areas, and embryos in these droplets stopped growing and eventually died if not transferred promptly. During microinjection, ideal outcomes were achieved when the needle penetrated the membrane without resistance and embryos maintained a spherical morphology with no leakage.

Embryos that retained a spherical morphology had a higher survival rate, whereas damaged embryos showed deformation or loss of cytoplasmic content. Through the adoption of these protocols, we can dissect the molecular pathways involved in development and regeneration of complex sensory organs in apple snails. Through our findings, we established apple snails as a novel system to answer questions about development, regeneration, evolution, and molluscan physiology.

Our results highlight the conserved roles of genes between apple snails and vertebrae animals, opening the door to questions about visual system regeneration and evolution.