Cells and Shells: New Research into Early Oyster Development Gives Insight into Potential Sterility Methods

There are many reasons why oyster farmers don't want their shellfish to reproduce. Sterile oysters can grow faster and meatier because they don't dedicate any energy to reproduction. Sterile oysters also limit the potential to create wild populations, which helps protect native wildlife and habitats.

Today, many oyster farms purchase triploid oyster "seed" from land-based hatcheries. Triploid oysters have an extra set of chromosomes that makes them functionally sterile, but also potentially more vulnerable to stressful conditions like warming oceans and disease.

A new study sheds light on early oyster development and could pave the way for new oyster sterility induction techniques. It could be an alternative to the more-vulnerable triploids currently used by industry. The study was conducted by NOAA Fisheries Northwest Fisheries Science Center and collaborators at the University of Washington.

"The alternative sterility method we're investigating has shown great promise in finfish, but is facing a major obstacle in shellfish. This study allows us to overcome this obstacle by identifying genes involved in the formation of reproductive cells," said Mackenzie Gavery, Ph.D. with NOAA's Northwest Fisheries Science Center, who led this study.

Primordial germ cells are the precursors of reproductive cells, or gametes. The goal of this study was to identify genes that are uniquely expressed in the primordial germ cells of oysters. NOAA scientists and partners successfully identified a suite of candidate genes that show promise for their role in primordial germ cell development.

Understanding how primordial germ cells are formed and develop into mature reproductive cells could help scientists develop methods to stop oyster's reproductive cells from developing in the first place.

The Early Life of an Oyster

Oysters are broadcast spawners, releasing eggs and sperm into the water column. When an egg is fertilized it becomes an embryo, its cells dividing and multiplying. It first becomes a free-swimming larva, then eventually settles as a tiny shelled oyster.

The genes expressed in these multiplying cells provide information about their pathways of development-what kinds of cells they'll eventually become.

Genes that are only expressed in a single cell type can serve as flags. Researchers can use these "gene expression markers" to better understand what controls the development, and differentiation, of particular cell types and tissues.

Prior to this study, not much was known about the genes expressed in oyster primordial germ cells. Because they don't copy themselves much (to prevent genetic mutations to the oyster's important reproductive development), they represent a very rare cell type in an embryo. "This makes oyster primordial germ cells difficult to study-it's like finding a needle in a haystack," said Gavery. "Finding them calls for specialized research techniques."

Searching for Gene Expression Markers

To identify the gene expression markers of primordial germ cells, NOAA scientists used single-cell RNA sequencing to analyze Pacific oyster embryos as they developed.

Traditional gene expression sequencing scrambles together the cells of a sample, producing an average gene expression. In contrast, single-cell RNA sequencing isolates the information in each individual cell, creating a detailed profile of what genes are expressed in different kinds of cells.

Using oyster parents, or brood stock, donated by Taylor Shellfish Hatchery in Washington, scientists mixed together oyster eggs and sperm in seawater to fertilize the eggs. Oyster embryos develop quickly, so the scientists had to keep a close eye on them under a microscope. Every 35 minutes they moved some of the embryos to the refrigerator to pause development for a short time. This resulted in a batch of oyster embryos at each stage of cellular division.

Next, the scientists prepared and genetically sequenced the oysters. Each embryo produces a wealth of gene expression data. For example, of the more than 18,500 cells sequenced from later stage embryos, each cell expressed an average of 2,828 genes.

This abundance of information allowed NOAA scientists and their collaborators to map gene expression trends across the stages of oyster development. They could then identify gene expression markers that indicate what those cells may become.

Identifying Shellfish Cells

Through this process, NOAA scientists discovered that the earliest stages of oysters show few distinct cell clusters with the gene expression markers of becoming primordial germ cells. However, roughly 10 hours after fertilization, later stage embryos show clear gene expression markers for cells that will become primordial germ cells.

Encouragingly, many of the genes expressed in oyster primordial germ cells have been associated with the development of reproductive cells in other animals, too--like the California mussel.

In addition to identifying genes associated with primordial germ cells, this investigation into oyster development also yielded new insights about which cells will become:

• Larval muscle
• The shell
• Other parts of the oyster

Paving the Way for New Aquaculture Techniques

These discoveries all have potential applications for the aquaculture industry. Better understanding which cells will become oyster shells could be used to help breed harder oysters that are more resilient to ocean acidification. And by mapping the gene expression markers that signal a cell's intention to become a primordial germ cell, we are better equipped to advance a novel sterility approach for oysters.

This approach, using small molecules to temporarily "silence" the expression of a key gene, essentially blocks primordial germ cell formation in embryos. Without primordial germ cells, they don't develop reproductive cells, and they're sterile. Since the genes are only temporarily silenced, this technique isn't genetic modification.

"Identifying these genes was critically important because we can't rely on what is known in other animals, like other kinds of invertebrates or fish, because mollusks are so unique!" said Gavery. Although it's yet to be tested for oysters, this method has shown success in fish. In a recent study, Adam Luckenbach, Ph.D., another researcher at Northwest Fisheries Science Center, introduced this approach to produce sterile sablefish for aquaculture .

By continuing to advance this methodology in shellfish, researchers could provide the same benefits of triploid oysters to the aquaculture industry, without compromising the oyster's hardiness. This work underscores NOAA's commitment to providing scientific solutions that benefit seafood farmers-promoting a resilient and robust domestic aquaculture industry.