Name: The Golden Apple Snail Pomacea canaliculata: From Zygotes to Stable Mutant Lines (Video) | JoVE Journal
Uploaded: 2025-12-23T14:00:00.000+00:00
Duration: 10 min 8 s
Description: Learn to generate stable mutant lines in Pomacea canaliculata using CRISPR/Cas9 microinjection and embryo culture. This protocol enables gene function studies in apple snails for developmental biology research.

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5 minutes

We would like to thank Dr. Julia Peloggia de Castro and the Accorsi Lab members for their critical reading of the manuscript. We gratefully acknowledge Dr. Alejandro S&#225;nchez Alvarado and the S&#225;nchez Alvarado Lab at the Stowers Institute for Medical Research (Kansas City, MO) for supporting the development of these protocols. We also thank the UC Davis Student Housing and Dining Services for sourcing and supplying organic lettuce for our colony of apple snails. This work was supported by start-up funding (Dr. Alice Accorsi) and the UC Davis Award for Innovation and Creative Vision (Dr. Alice Accorsi).

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<li>Perry, K. J., Henry, J. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CRISPR%2FCas9-mediated+genome+modification+in+the+mollusc%2C+Crepidula+fornicata%5BTitle%5D)+AND+%22Genesis%22%5BJournal%5D)">CRISPR/Cas9-mediated genome modification in the mollusc, Crepidula fornicata.</a> Genesis. 53 (1), 237-244 (2015).</li>
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<li>Yu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeted+gene+disruption+in+Pacific+oyster+based+on+CRISPR%2FCas9+ribonucleoprotein+complexes%5BTitle%5D)+AND+%22Mar+Biotechnol%22%5BJournal%5D)">Targeted gene disruption in Pacific oyster based on CRISPR/Cas9 ribonucleoprotein complexes.</a> Mar Biotechnol. 21 (1), 301-309 (2019).</li>
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<li>Bono, J. M., Olesnicky, E. C., Matzkin, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Connecting+genotypes%2C+phenotypes+and+fitness%3A+Harnessing+the+power+of+CRISPR%2FCas9+genome+editing%5BTitle%5D)+AND+%22Mol+Ecol%22%5BJournal%5D)">Connecting genotypes, phenotypes and fitness: Harnessing the power of CRISPR/Cas9 genome editing.</a> Mol Ecol. 24 (1), 3810-3822 (2015).</li>
<li>Hayes, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Insights+from+an+integrated+view+of+the+biology+of+apple+snails+%28Caenogastropoda%3A+Ampullariidae%29%5BTitle%5D)+AND+%22Malacologia%22%5BJournal%5D)">Insights from an integrated view of the biology of apple snails (Caenogastropoda: Ampullariidae).</a> Malacologia. 58 (1), 245-302 (2015).</li>
<li>Estebenet, A. L., Martín, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pomacea+canaliculata+%28Gastropoda%3A+Ampullariidae%29%3A+Life-history+traits+and+their+plasticity%5BTitle%5D)+AND+%22Biocell%22%5BJournal%5D)">Pomacea canaliculata (Gastropoda: Ampullariidae): Life-history traits and their plasticity.</a> Biocell. 26 (1), 83-89 (2002).</li>
<li>Pasquevich, M. Y., Dreon, M. S., Heras, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+major+egg+reserve+protein+from+the+invasive+apple+snail+Pomacea+maculata+is+a+complex+carotenoprotein+related+to+those+of+Pomacea+canaliculata+and+Pomacea+scalaris%5BTitle%5D)+AND+%22Comp+Biochem+Physiol+B+Biochem+Mol+Biol%22%5BJournal%5D)">The major egg reserve protein from the invasive apple snail Pomacea maculata is a complex carotenoprotein related to those of Pomacea canaliculata and Pomacea scalaris.</a> Comp Biochem Physiol B Biochem Mol Biol. 169 (1), 63-71 (2014).</li>
<li>Giraud-Billoud, M., Gamarra-Luques, C., Castro-Vazquez, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+anatomy+of+male+copulatory+organs+of+Pomacea+canaliculata+%28Caenogastropoda%2C+Ampullariidae%29%5BTitle%5D)+AND+%22Zoomorphology%22%5BJournal%5D)">Functional anatomy of male copulatory organs of Pomacea canaliculata (Caenogastropoda, Ampullariidae).</a> Zoomorphology. 132 (1), 129-143 (2013).</li>
<li>Mu, H., Sun, J., Heras, H., Chu, K. H., Qiu, J. -W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dataset+for+the+proteomic+and+transcriptomic+analyses+of+perivitelline+fluid+proteins+in+Pomacea+snail+eggs%5BTitle%5D)+AND+%22Data+Brief%22%5BJournal%5D)">Dataset for the proteomic and transcriptomic analyses of perivitelline fluid proteins in Pomacea snail eggs.</a> Data Brief. 15 (1), 203-207 (2017).</li>
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<li>Seuffert, M. E., Martín, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Juvenile+growth+and+survival+of+the+apple+snail+Pomacea+canaliculata+%28Caenogastropoda%3A+Ampullariidae%29+reared+at+different+constant+temperatures%5BTitle%5D)+AND+%22Springerplus%22%5BJournal%5D)">Juvenile growth and survival of the apple snail Pomacea canaliculata (Caenogastropoda: Ampullariidae) reared at different constant temperatures.</a> Springerplus. 2 (1), 312(2013).</li>
<li>McKenna, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Whole-organism+lineage+tracing+by+combinatorial+and+cumulative+genome+editing%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Whole-organism lineage tracing by combinatorial and cumulative genome editing.</a> Science. 353 (1), aaf7907(2016).</li>
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</ol>

The authors have no conflict of interest to declare.

This series of protocols provides a comprehensive framework for the generation and maintenance of P. canaliculata mutant lines, providing the scientific community with a new genetically tractable invertebrate model6. Now, this animal can be leveraged to significantly advance the studies in several fields beyond the camera-type eye regeneration one, such as comparative spiralian embryology, environmental toxicology, and comparative molluscan physiology. The methodology integrates classic g...

The golden apple snail, Pomacea canaliculata, is emerging as a valuable model organism in regenerative biology due to its remarkable ability to regenerate complex organs, including its camera-type eyes, after complete amputation1 (Figure 1A). P. canaliculata&#8217;s camera-type eyes, comprising a retina with photoreceptors, a cornea, and a lens organized in a large single chamber, closely resemble vertebrate eyes, making it an ideal and almost unique system for investigating the molecular underpinnings of this complex sensory organ, complete regeneration1,2,3,4,5,6. In addition to these incredible biological features, its diploid genome, genetic tractability, direct development (lack of free-living larval stage and metamorphosis), number of embryos available daily, and ease of laboratory maintenance enhance its suitability for molecular and cellular research6,7,8,9,10 (Figure 1B,C).
Stable mutant lines offer a powerful means to probe gene function. The CRISPR/Cas9 system enables easier genetic manipulation of new species of interest11,12,13,14,15,16,17. Some major challenges remain when applying CRISPR/Cas9 mutagenesis to a new species, including obtaining one-cell stage embryos, performing injections without compromising their viability, and raising the injected animals to sexual maturity. All these methods in P. canaliculata have been recently developed from scratch, significantly increasing the potential of this new system to study the genetic basis of camera-type eye development, regeneration, and evolution2,3,6,18. Genome editing and germline transmission are the first steps to the development of transgenic reporter lines and conditional mutants19. Such protocols are both timely and essential, given the relative novelty of applying these techniques to mollusks and the interesting biological features of P. canaliculata. These tools will provide essential resources for researchers studying P. canaliculata and for those aiming to apply similar methodologies to species with similar reproductive or developmental traits.
P. canaliculata populations consist of distinct males and females20,21&#160;(Figure 1D,E). After mating, the females store the sperm to internally fertilize the oocytes shortly before laying the zygotes20. Apple snails lay large clutches, each containing about 100&#8211;300 embryos20,21 (Figure 1B). Each embryo is housed in a capsule that has a hard outer casing and is filled with pink perivitelline fluid (PVF)22. The PVF provides key nutrients and protection to the developing embryo, while the outer layer prevents dehydration22. Capsules are laid outside the water, and they go through important changes during the first 24 h; freshly laid eggs transition from a soft external membrane and a dense and opaque PVF to a harder casing and a less dense, more transparent PVF20 (Figure 1B&#160;and&#160;Figure 2). Zygotes go through the first cell division approximately 6 h post fertilization (hpf), providing ample time for the microinjections6.
This study presents all the methods needed to generate stable mutant lines in P. canaliculata. The pipeline includes maintenance of an apple snail colony, efficient collection of healthy zygotes, and their microinjection using an inverted microscope equipped with micromanipulators and a MICRO-ePORE Pinpoint Cell Penetrator. This work further describes&#160;an innovative procedure, embryo ex ovo culture, for culturing embryos outside their capsules. Central to this is the isolation of the PVF, the natural environment for apple snail embryonic development, and its quality control for successful ex ovo embryo culture. Finally, methods for growing and genotyping apple snails, as well as a guideline for designing the crossing for long-term maintenance of the mutant lines, are provided.
By establishing and detailing these protocols, the groundwork is laid for generating stable mutant lines in apple snails, opening avenues for genetic studies in a new, yet highly promising, invertebrate system. At present, the application of genetic manipulation to this model stands to significantly advance the understanding of sensory system development and regeneration. Considering this is one of the very few mollusks where stable mutant lines can be established to date11,14,15,16,17, the vision is that this system will be adopted in other biological fields to study their physiology and specializations.&#160;

<table><tbody><tr><td>1.7 mL microcentrifuge tubes</td><td>VWR</td><td>87003-294</td><td></td></tr><tr><td>15 mL tubes</td><td>VWR</td><td>21008-670</td><td></td></tr><tr><td>2 mL microcentrifuge tubes&amp;nbsp;</td><td>VWR</td><td>87003-298</td><td></td></tr><tr><td>4-well plates</td><td>Fisher Scientific</td><td>FB012926</td><td></td></tr><tr><td>50 mL&amp;nbsp; tubes</td><td>VWR</td><td>21008-690</td><td></td></tr><tr><td>6-well plates</td><td>Fisher Scientific</td><td>89093-494</td><td></td></tr><tr><td>96-well plate for qPCR</td><td>BioRad</td><td>HSP9601</td><td></td></tr><tr><td>Agarose</td><td>Fisher Scientific</td><td>BP1356500</td><td></td></tr><tr><td>Calcion-P</td><td>Brightwell Aquatics</td><td>N/A</td><td></td></tr><tr><td>Calcium chloride dihydrate</td><td>Research Products International</td><td>C36200</td><td></td></tr><tr><td>Capillaries with filament - Borosilicate&amp;nbsp;</td><td>Harvard Apparatus</td><td>30-0038</td><td></td></tr><tr><td>Capillaries without filament - Borosilicate&amp;nbsp;</td><td>Harvard Apparatus</td><td>30-0016</td><td></td></tr><tr><td>Cas9 protein HiFi V3</td><td>IDT</td><td>1081060</td><td></td></tr><tr><td>CellTram 4r Air</td><td>Calibre Scientific</td><td>EPE-5196000013</td><td></td></tr><tr><td>CFX Maestro Software for qPCR</td><td>BioRad</td><td>12013758</td><td></td></tr><tr><td>Chiller for microscope&amp;nbsp;</td><td>Linkam</td><td>T96 PE100-ZAL System&amp;nbsp;</td><td></td></tr><tr><td>Depression slide&amp;nbsp;</td><td>Fisher Scientific</td><td>S00376</td><td></td></tr><tr><td>Dextran</td><td>Fisher Scientific</td><td>D3329</td><td></td></tr><tr><td>DNA Ladder 1kb</td><td>Promega&amp;nbsp; Corporation</td><td>G5711</td><td></td></tr><tr><td>Electrophoresis chamber and gel caster</td><td>BioRad</td><td>1704469</td><td></td></tr><tr><td>Ethanol</td><td>Koptec</td><td>UN1170</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS) - heat inactivated</td><td>Fisher Scientific</td><td>A5670801</td><td></td></tr><tr><td>Filtered Tips P1000</td><td>Rainin&amp;nbsp;</td><td>30389272</td><td></td></tr><tr><td>Filtered Tips P20/P2</td><td>Rainin&amp;nbsp;</td><td>30389274</td><td></td></tr><tr><td>Filtered Tips P200</td><td>Rainin&amp;nbsp;</td><td>30389276</td><td></td></tr><tr><td>Galactose&amp;nbsp;</td><td>Fisher Scientific</td><td>BP656500</td><td></td></tr><tr><td>GoTaq(R) Hot Start&amp;nbsp;</td><td>Promega&amp;nbsp; Corporation</td><td>M5123</td><td></td></tr><tr><td>gRNA</td><td>IDT</td><td>N/A</td><td></td></tr><tr><td>Hemostat locking scissor clamp</td><td>Amazon</td><td>DS01KEL</td><td></td></tr><tr><td>HEPES</td><td>Affymetrix</td><td>16926</td><td></td></tr><tr><td>Incubator DigiTherm Heating/Cooling</td><td>Tritech Research</td><td>DT2-MP-47L</td><td></td></tr><tr><td>Instant Ocean Sea Salt&amp;nbsp;</td><td>Fisher Scientific</td><td>S81800</td><td></td></tr><tr><td>Inverted Microscope with 20x DIC objective</td><td>Zeiss&amp;nbsp;</td><td>AxioObserver</td><td></td></tr><tr><td>L-cysteine</td><td>Millipore Sigma</td><td>C7352</td><td></td></tr><tr><td>Lettuce, organic</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>Loading dye GelRed</td><td>Biotum</td><td>41011</td><td></td></tr><tr><td>Magnesion-P</td><td>Brightwell Aquatics</td><td>N/A</td><td></td></tr><tr><td>Magnesium chloride</td><td>Sigma</td><td>M0250</td><td></td></tr><tr><td>Microcentrifuge - refrigerated&amp;nbsp;</td><td>Eppendorf</td><td>5425R</td><td></td></tr><tr><td>MICRO-ePORE pinpoint cell penetrator&amp;nbsp;</td><td>World Precision Instruments</td><td>300684</td><td></td></tr><tr><td>Microforge</td><td>Narishige</td><td>MF-900</td><td></td></tr><tr><td>Microinjector FemtoJet 4i</td><td>Calibre Scientific</td><td>5252000021</td><td></td></tr><tr><td>Microloader tapered tip</td><td>Calibre Scientific</td><td>930001007</td><td></td></tr><tr><td>Micromanipulators TransferMan 4r&amp;nbsp;</td><td>Calibre Scientific</td><td>5193000020</td><td></td></tr><tr><td>Needle puller</td><td>Sutter Instrument Company</td><td>P-97</td><td></td></tr><tr><td>NP-40</td><td>Fisher Scientific</td><td>28324</td><td></td></tr><tr><td>Nuclease free water</td><td>VWR</td><td>E476</td><td></td></tr><tr><td>Paraffin oil</td><td>Millipore Sigma</td><td>PX0047</td><td></td></tr><tr><td>PCR Strip Tubes</td><td>VWR</td><td>490003-706</td><td></td></tr><tr><td>Petri dishes 100 mm</td><td>VWR</td><td>25373-100</td><td></td></tr><tr><td>Petri dishes 35mm</td><td>VWR</td><td>25373-041</td><td></td></tr><tr><td>Petri dishes 60mm</td><td>VWR</td><td>25373-085</td><td></td></tr><tr><td>Phenol red</td><td>Millipore Sigma</td><td>P0290</td><td></td></tr><tr><td>Pipettes P2</td><td>Rainin&amp;nbsp;</td><td>17014393</td><td></td></tr><tr><td>Pipettes P20</td><td>Rainin&amp;nbsp;</td><td>17014392</td><td></td></tr><tr><td>Pipettes P200</td><td>Rainin&amp;nbsp;</td><td>17014391</td><td></td></tr><tr><td>Pipttes P1000</td><td>Rainin&amp;nbsp;</td><td>17014382</td><td></td></tr><tr><td>Plate Sealing Film for qPCR</td><td>BioRad</td><td>17010701</td><td></td></tr><tr><td>Potassium chloride</td><td>Fisher Scientific</td><td>P217</td><td></td></tr><tr><td>Power Supply PowerPac</td><td>BioRad</td><td>1645050</td><td></td></tr><tr><td>Primers</td><td>IDT</td><td>N/A</td><td></td></tr><tr><td>Proteinase K</td><td>Millipore Sigma</td><td>P4850</td><td></td></tr><tr><td>qPCR machine</td><td>BioRad</td><td>CFX96</td><td></td></tr><tr><td>qPCR probes</td><td>IDT</td><td>N/A</td><td></td></tr><tr><td>Rift Lake dKH</td><td>Brightwell Aquatics</td><td>N/A</td><td></td></tr><tr><td>Scissors for microdissection</td><td>Fine Science Tools</td><td>15006-09</td><td></td></tr><tr><td>Sequencing Service</td><td>Genewiz</td><td>N/A</td><td></td></tr><tr><td>Siphon Water Changer</td><td>Amazon</td><td>11278605</td><td></td></tr><tr><td>Snail tank lids - 18 qt</td><td>Cambro&amp;nbsp;</td><td>SFC12</td><td></td></tr><tr><td>Snail tank lids - 8 qt and 6 qt</td><td>Cambro&amp;nbsp;</td><td>SFC6</td><td></td></tr><tr><td>Snail tanks - 18 qt</td><td>Cambro&amp;nbsp;</td><td>18SFSCW</td><td></td></tr><tr><td>Snail tanks - 6 qt</td><td>Cambro&amp;nbsp;</td><td>6SFSCW</td><td></td></tr><tr><td>Snail tanks - 8 qt</td><td>Cambro&amp;nbsp;</td><td>8SFSCW</td><td></td></tr><tr><td>Sodium chloride</td><td>Fisher Scientific</td><td>S271500</td><td></td></tr><tr><td>Sodium hydroxyde</td><td>Fisher Scientific</td><td>SS2551</td><td></td></tr><tr><td>Sodium hypochlorite&amp;nbsp;</td><td>Clorox (Bleach)</td><td>N/A</td><td></td></tr><tr><td>Stereo microscope with fluorescent light</td><td>Zeiss</td><td>Discovery V8</td><td></td></tr><tr><td>Stereo microscope with transmitted light (mirror base)</td><td>Zeiss</td><td>Stemi 508</td><td></td></tr><tr><td>SYBR Green Supermix for qPCR</td><td>BioRad</td><td>1725270</td><td></td></tr><tr><td>TAE buffer</td><td>VWR</td><td>76371-722</td><td></td></tr><tr><td>Thermal cycler</td><td>Eppendorf</td><td>Mastercycler x40</td><td></td></tr><tr><td>Tips P1000</td><td>Rainin&amp;nbsp;</td><td>30389215</td><td></td></tr><tr><td>Tips P20/P2</td><td>Rainin&amp;nbsp;</td><td>30389228</td><td></td></tr><tr><td>Tips P200</td><td>Rainin&amp;nbsp;</td><td>30389245</td><td></td></tr><tr><td>Tris pH 8</td><td>VWR</td><td>10128-396</td><td></td></tr><tr><td>Tween 20</td><td>Sigma Aldrich</td><td>P1379</td><td></td></tr><tr><td>Tweezers with curved end</td><td>A2Z SciLab</td><td>686797862535</td><td></td></tr><tr><td>Water Recirculation System - Custom-made</td><td>Iwaki America</td><td>N/A</td><td></td></tr></tbody></table>

the golden apple snail pomacea canaliculata: from zygotes to stable mutant lines

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

The freshwater golden apple snail (Pomacea canaliculata) is emerging as a powerful model organism for regenerative biology, particularly for studying the molecular mechanisms of sensory organ regeneration. They can fully regenerate their camera-type eyes, which have vertebrate-like anatomical features such as retina, cornea, and lens organized in a closed chamber. Coupled with a relatively small diploid genome, genetic tractability, and ease of laboratory maintenance, P. canaliculata offers unique advantages for studies beyond developmental and regenerative biology. This paper presents a comprehensive workflow for generating and maintaining apple snail mutant lines, making these animals one of the very few genetically tractable mollusks available. The possibility of knocking out genes of interest opens the door to understanding the genetic and molecular basis of sensory organ development and regeneration, as well as other aspects of molluscan biology. Here, we provide detailed protocols for the effective maintenance of a large apple snail colony, collection and microinjection of their zygotes, the use of perivitelline fluid for successful ex ovo culture, and techniques for genotyping and maintaining stable mutant lines. By enabling targeted genetic manipulation in this novel model and disseminating methods for functional genomics in mollusks, we aim to encourage the community to grow and adopt apple snails for further studies across biological fields.

The possible outcomes of the ex ovo culture protocol are illustrated in Figure 5. To test the quality of the ePVF, 2 dpf embryos were isolated and cultured in droplets of ePVF, overlaid with paraffin oil (Figure 4). Viable embryos maintain a spherical morphology and clarity of tissues, and they grow with significant changes in their morphology every 24 h. Successful culture is represented by embryonic growth and&#160;the formation of some air bubbles on...

This study developed a workflow for the generation and maintenance of stable mutant lines for the freshwater apple snail, Pomacea canaliculata. The protocols include the collection of zygotes, their microinjection with the CRISPR/Cas9 system components to induce genome editing, ex ovo culture of embryos, and genotyping after controlled crosses.

The freshwater golden apple snail (Pomacea canaliculata) is emerging as a powerful model organism for regenerative biology, particularly for studying the ...

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.

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

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