Morpholino oligomer delivery via bath immersion for use in reverse genetic studies on the early development of eastern oysters (Crassostrea virginica)

Abstract

The eastern oyster genome and expanding omics data have provided valuable insights into this species. However, the limited availability of molecular toolboxes constrains the further exploration of gene functional investigation. In this study, we applied an emerging immersion-based gene silencing technology and developed a detailed protocol for delivering Morpholino oligomer (MO) to eastern oysters. First, two target genes, cv-gcl and cv-vasa, were cloned, and their expressions in ovaries and during embryogenesis were characterized. Both genes were maternally deposited to oocytes, and the expression of cv-vasa decreased steadily after fertilization, while cv-gcl peaked around 20 hours post-fertilization. The post-fertilization immersion treatment was more effective (three- to four-fold) than the pre-fertilization treatment, indicating a stronger MO uptake after fertilization. MOs designed against these two genes were administered via Vivo using bath immersion treatment following fertilization. There was no difference in survival rates (D-larvae yield) at 1 day post-fertilization (dpf) when treating embryos with cvgcl-MO-Vivo up to 20 μM, while deleterious effects started to emerge when increasing the concentration to 30 and 40 μM. The cvvasa-MO-Vivo treated group exhibited a lower Vasa level compared to the control-MO-Vivo group, suggesting the potential knockdown of the target gene. Most importantly, we achieved real-time visualization of MO uptake by conjugating fluorescence-labeled MO with a cell-penetrating peptide. Monitoring fluorescent intensity inside oyster larvae revealed that MO delivery occurred during early embryogenesis, and the signal retained up to 6 dpf. The immersion and fluorescence-traceable approach described here is a highly efficient reverse genetic method of delivering and monitoring MO in a large number of developing eastern oyster embryos to study the function of genes of interest involved in early development.