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.