This post describes previous protocols for PolyIC immune priming in oysters using emmersion/bath methods. We will use these protocols to design PolyIC exposure in oysters at Point Whitney.

Overview

Poly(I:C) is a non-infectious elicitor and viral mimic in the form of synthetic double stranded RNA (dsRNA). Poly(I:C) is synthetic, double-stranded RNA molecule polyribonucleotide that has been widely used to induce a type I interferon (IFN) response in many species.

In oysters, it has been shown to produce a protective immune response. For example:

• Lafont et al. 2023
• Green et al. 2013
• Martins et al. 2020
• Lorgeril et al. 2018

Previous protocols in the Roberts Lab

Matt George has done some previous work with PolyIC using injection methods in the lab before. He did some tests to look at water bath vs injection protocols detailed in his notebook in these posts: Part 1, Part 2, and Part 3.

In his tests, Matt did the following:

“10 diploid and 10 triploid oysters (naive) were added to an emersion bath containing 500 ml of seawater contianing 10 ug/ml Poly(I:C), with a bubbler. Lafont et al 2017 found that emersion at 38 ug/ml worked for 24 hrs, so we will see.”

Matt didn’t have any follow up in his notebook, so I’ll reach out to see what the results were.

Water bath/emmersion protocols in the literature

There is one study I could find that has used emmersion protocols for PolyIC exposure.

Lafont et al. 2023 found the emmersion in PolyIC provided equal protection to injection methods, which are more commonly used. Injection includes anesthesia for oysters followed by direct injection into tissues. If a water bath with PolyIC works equally well, this would be a much easier protocol to use.

In one of their experiments, Lafont et al. (2023) exposed 20 oysters to either 0.76 ug per mL or 0.38 ug per mL in 100 mL of PolyIC in seawater for 2.5 hours. Oysters were then put in seawater for 24 hours to recover and then injected with pathogens (OsHV-1 or heat killed vibrio) to test immune response over 8 days.

They found that survival was higher with PolyIC regardless of dose than those without PolyIC exposure (~90% survival vs ~50% survival without priming). This was similar to survival observed with a high does of injected PolyIC (~85% survival).

Suggested protocol for us to try

Based on Matt’s tests and the study outlined above, I think we should try a PolyIC bath/emmersion at the following parameters:

• 0.38 ug/mL concentration of PolyIC in a seawater solution
• 2.5 hour emmersion in seawater solution

I haven’t used PolyIC before, so we need to look at manufacturers instructions for mixing the solutions properly.