Direct Setting of Eastern Oyster (Crassostrea virginica) Larvae Confirmed with Calcein, a Fluorochrome Dye

Ongoing efforts to restore eastern oyster ( Crassostrea virginica ) populations in regions of low natural recruitment rely on stocking of juveniles (spat on shell) to rebuild populations. Often, remote setting is used, entailing releasing hatchery-produced larvae into recirculating tanks filled with oyster shells. Other restoration methods that rely on releasing C. virginica larvae directly onto the shell in situ, called “direct setting,” have shown promise but were unable to definitively prove larval origin without the use of enclosures. The objective of this study was to determine if tagging C. virginica with calcein, a fluorochrome dye, could be a viable method for confirming larval origin in studies of direct setting in Chesapeake Bay. To do so, C. virginica larvae conditioned in water from adult C. virginica were marked with calcein and released by divers directly onto three 3.6-m 2 research sites constructed of oyster shell bags during July 2019 and September 2019 and recovered after 7 days. All shell bags were moved to flow-through tanks on land and spat on a subsample of valves were counted in each bag 8–12 days after deployment. Spat on the remaining valves were counted 42 to 46 days post deployment. A total of 119,020 spat were found on 84 shell bags from the two deployments during the initial settlement counts conducted just after shell bags were recovered. All recovered juveniles that were viewed under blue light excitation ( n = 84) contained the calcein tag, indicating that these spat were derived from larvae released over the reefs. Initial settlement efficiencies on the sites ranged from 0.1 to 3.4% in July and September, respectively. The salinities experienced in July were below average and may have contributed to reduced larval survival compared to that in September. Shell bags contained zero to 90 spat per shell. Spat settlement was greatest closest to where the larvae were released (87% of spat were found in 12% of bags; the high-count bags were clustered around the larval release locations). Overall, 6 shell bags out of 190 deployed had spat per shell estimates similar to remote larval setting (hatchery) targets (10–20 spat per shell) and 6 had spat per shell values higher than hatchery targets. The presence of the calcein mark in recovered spat confirmed larval origin, and together with the observed setting efficiencies suggests there is promise for developing remote larval setting as a stock enhancement technique. However, more work