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The goal of this study was to evaluate the efficacy of phenotypic traits and two genetic assays to distinguish between Mercenaria mercenaria, an important aquaculture species, and Mercenaria campechiensis, a conspecific species that is partially sympatric, but that has not been successfully farmed. The objectives were to evaluate (1) four established diagnostic phenotypic traits (lunule shape, thickness of concentric shell ridges, prominence of anterior side of the concentric ridges, and presence of purple nacre inside of the shells) to distinguish between species, (2) size polymorphism of PCR fragments from four genes [16S rRNA, 18S rRNA, Internal Transcribed Spacer genes (ITS1 and ITS2)], and (3) PCR-restriction fragment length polymorphism (RFLP) on three of these genes (16S, ITS1, and ITS2). Phenotypic traits correctly distinguished two geographically distant conspecific populations (two locations in Maine for M. mercenaria and in Bradenton, FL, for M. campechiensis); however, among all other locations, both species had overlapping phenotypic trait values. Although PCR fragments were obtained for all genes tested, size polymorphism was not identified between the two species. Restriction enzyme digestion of PCR fragments resulted in diagnostic fragments distinguishing the two species for 16S, ITS1, and ITS2. Although phenotypic scores failed to delineate between the two species in most locations, PCR-RFLP results failed to identify hybrids in wild or hatchery samples. Further investigation is needed to apply the PCR-RFLP assays on known hybrid offspring from crosses of M. mercenaria and M. campechiensis. Results have elucidated the difficulties in confidently detecting hybridization in quahogs; however, determining effective methods for species identification and hybrid detection will be important to address issues such as the prevalence and distribution of hybridization in aquaculture and in wild populations, and to determin the presence of M. campechiensis which are currently difficult to find.

Cryopreservation can preserve genetic materials in perpetuity and can be applied to oyster culture for breeding programs. Protocols exist for sperm cryopreservation in the eastern oyster Crassostrea virginica, but nonlethal sample collection is needed for valuable individuals, such as tetraploids, or specific lines. The goal of this study was to develop nonlethal methods for sperm collection in the eastern oyster. The objectives were (1) to evaluate natural spawning as a collection method, (2) to evaluate anesthesia methods to induce shell opening for biopsy, (3) to evaluate mechanical notching for biopsy, and (4) to verify notching combined with biopsy for collection and cryopreservation. Five males (of 60 oysters) spawned naturally after 7 h, with an average sperm concentration of 1.9 ± 1.0310 3 cells/mL (in 2 L seawater). No oysters (n ¼ 30) responded by opening during 36 h of treatment with 5% Dead Sea salt (containing 33.3% MgCl2), and 22 oysters (of 30) opened during the 36-h treatment with 5% Epson salt (MgSO4). Sperm collected by biopsy had fresh motility of 3%-80% and postthaw motility of 1%-5%; sperm production was 4.53 10 5 to 2.3310 8 cells per male. Mechanical notching did not cause mortality to oysters (n ¼ 20). After notching and biopsy with 18-G and 20-G needles, survival was 80% (16 of 20 for each). Sperm production was 5.42 3 10 7 cells by 18-G needle (n ¼ 8) with fresh motility of 16± 12% and postthaw motility of 3± 2%, and 1.35310 8 cells by 20-G needle (n ¼ 9) with fresh motility of 21± 20% and postthaw motility of 5 ± 4%. No differences were observed between samples biopsied with the 2 needle sizes (P $ 0.074). To verify notching and biopsy for nonlethal sperm collection, a total of 39 oysters were sampled to obtain 20 males, which averaged 99.48 ± 23.17 g total weight, 74.1 ± 6.0 mm shell height, and 60.9 ± 7.2 mm shell length. The sperm production was 3.6 ± 2.1 3 10 8 cells per male. Biopsied sperm showed 23 ± 12% fresh motility, 13 ± 6% postequilibration motility (after equilibration with 10% of DMSO for 30-60 min before freezing), and 6± 4% postthaw motility. Flow cytometry analysis indicated an average of 84± 4% of cells with intact plasma membranes for fresh sperm, and 59± 9% for postthaw sperm. Fertilization by thawed sperm averaged 20± 22% (from 1%-87%). No significant differences were observed between the biopsied samples and the dissected samples (lethal collection) for fresh motility (P ¼ 0.550), postequilibration motility (P ¼ 1.000), postthaw motility (P ¼ 0.101), fresh membrane integrity (P ¼ 1.000), or postthaw membrane integrity (P ¼ 1.000), but a difference was observed in fertilization (P ¼ 0.039; biopsied samples, 20± 22%; dissected samples, 68 ± 40%). Overall, this study developed notching combined with biopsy for sperm collection and cryopreservation in Eastern oysters that can be applied to valuable individuals and breeding programs.