Ami E. Wilbur

Strong seasonality of Bonamia sp. infection and induced Crassostrea ariakensis mortality in Bogue and Masonboro Sounds, North Carolina, USA

Asian oyster Crassostrea ariakensis is being considered for introduction to Atlantic coastal waters of the USA. Successful aquaculture of this species will depend partly on mitigating impacts by Bonamia sp., a parasite that has caused high C. ariakensis mortality south of Virginia. To better understand the biology of this parasite and identify strategies for management, we evaluated its seasonal pattern of infection in C. ariakensis at two North Carolina, USA, locations in 2005. Small (<50 mm) triploid C. ariakensis were deployed to upwellers on Bogue Sound in late spring (May), summer (July), early fall (September), late fall (November), and early winter (December) 2005; and two field sites on Masonboro Sound in September 2005. Oyster growth and mortality were evaluated biweekly at Bogue Sound, and weekly at Masonboro, with Bonamia sp. prevalence evaluated using parasite-specific PCR. We used histology to confirm infections in PCR-positive oysters. Bonamia sp. appeared in the late spring Bogue Sound deployment when temperatures approached 25 degrees C, six weeks post-deployment. Summer- and early fall-deployed oysters displayed Bonamia sp. infections after 3-4 weeks. Bonamia sp. prevalences were 75% in Bogue Sound, and 60% in Masonboro. While oyster mortality reached 100% in late spring and summer deployments, early fall deployments showed reduced (17-82%) mortality. Late fall and early winter deployments, made at temperatures <20 degrees C, developed no Bonamia sp. infections at all. Seasonal Bonamia sp. cycling, therefore, is influenced greatly by temperature. Avoiding peak seasonal Bonamia sp. activity will be essential for culturing C. ariakensis in Bonamia sp.-enzootic waters.

A genetic assessment of bay scallop (Argopecten irradians) restoration efforts in Florida’s Gulf of Mexico Coastal Waters (USA)

AbstractAs part of a comprehensive bay scallop restoration plan in Florida, we implemented a genetic monitoring program to evaluate the impact of shellfish restoration. Restoration involved the deployment of hatchery-produced scallops in cages (the restoration stock), which created spawner aggregations in locations that exhibited low densities of wild scallops. The success of the restorations was evaluated by comparing the genetic composition of wild scallops before (pre-restoration samples) and after (assessment samples) each deployment. The effectiveness of this approach in determining the contribution of the restoration stock relied on a two-part mitochondrial DNA (mtDNA) assay developed to differentiate between scallops produced by the restoration stock and those produced by the remnant wild population. Assessment scallops were sequenced initially for a 417 base-pair fragment (segment 2), and if the mtDNA sequence was found to be identical to that of any restoration-stock scallop, the assessment scallop was sequenced for an additional 462 base pairs (segment 1). We screened assessment samples from six locations in west-central Florida for evidence of a significant contribution from the restoration stock to the wild population, manifested as a significant increase in the frequency of the haplotypes diagnostic of the restoration stock. In the 3 years of monitoring, 23 of 512, 13 of 600, and 19 of 991 assessment- sample scallops collected from the vicinities of the three restoration locations had haplotypes identical to those of restoration-stock individuals. In all years, the assessment-sample frequencies of haplotypes characteristic of the restoration stock were not significantly different from prerestoration-sample frequencies. This absence of a detectable contribution based on genetic data contrasts with abundance data from one location, which suggests a dramatic increase in the abundance of scallops following the restoration effort.

Natural and Anthropogenic Forces Shape the Population Genetics and Recent Evolutionary History of Eastern United States Bay Scallops (Argopecten irradians)

ABSTRACT Bay scallops (Argopecten irradians Lamarck) are ecologically important in U.S. Atlantic waters off northeastern states and in the Florida Gulf of Mexico, and have been intensely harvested from both of those regions for decades. However, a detailed study comparing their basic population genetic structures using more than a single type of genetic marker has not been conducted. Through such a study, key phylogeographic, taxonomic, and fisheries issues can be addressed. We used variation in allozyme loci and mitochondrial DNA restriction fragment length polymorphisms to evaluate and compare the population genetic structures of bay scallops from those two regions, to propose a new interpretation for the composition of the North Carolina bay scallop population, to resolve the taxonomic quandary of Argopecten irradians taylorae, and to evaluate the apparent and potential genetic effects of the common fishery practice of hatchery-based stock enhancement on the genetic diversity and relatedness of Atlantic bay scallop populations. Atlantic Ocean (North Carolina through New York) bay scallop populations are genetically more distant from each other than are Florida Gulf bay scallop populations, except those in Florida Bay. Each Atlantic population has a different phylogeographic history, is quasi-independent, and should be treated as a genetically unique entity. The North Carolina bay scallop population is composed of Argopecten irradians irradians individuals, but also has genetic input from Argopecten irradians concentricus. Bay scallops occurring in Florida Bay constitute a population of A. i. concentricus that has diverged from other Florida Gulf populations because it has undergone repeated contractions and expansions of varying magnitude and is nearly isolated from other bay scallop populations. For the common practice of hatchery-based stock enhancement in the Atlantic, broodstock bay scallops should be taken from the same genetic population, and all stock enhancement efforts should include comprehensive genetic monitoring programs. In some cases, improving the abundance and density of bay scallop aggregations through habitat improvement may be preferable to stock enhancement for bay scallop restoration, but in other cases genetically conscientious stock supplementation or restoration may be the only alternative.

Quantitative PCR assay to determine prevalence and intensity of MSX (Haplosporidium nelsoni) in North Carolina and Rhode Island oysters Crassostrea virginica.

The continuing challenges to the management of both wild and cultured eastern oyster Crassostrea virginica populations resulting from protozoan parasites has stimulated interest in the development of molecular assays for their detection and quantification. For Haplosporidium nelsoni, the causative agent of multinucleated sphere unknown (MSX) disease, diagnostic evaluations depend extensively on traditional but laborious histological approaches and more recently on rapid and sensitive (but not quantitative) end-point polymerase chain reaction (PCR) assays. Here, we describe the development and application of a quantitative PCR (qPCR) assay for H. nelsoni using an Applied Biosystems TaqMan® assay designed with minor groove binder (MGB) probes. The assay was highly sensitive, detecting as few as 20 copies of cloned target DNA. Histologically evaluated parasite density was significantly correlated with the quantification cycle (Cq), regardless of whether quantification was categorical (r2 = 0.696, p < 0.0001) or quantitative (r2 = 0.797, p < 0.0001). Application in field studies conducted in North Carolina, USA (7 locations), revealed widespread occurrence of the parasite with moderate to high intensities noted in some locations. In Rhode Island, USA, application of the assay on oysters from 2 locations resulted in no positives.

Analysis of Spatiotemporal Genetic Variability in Eastern Oyster Crassostrea virginica (Gmelin, 1791) Mtdna 16S Sequences Among North Carolina Populations

ABSTRACT Spatial and temporal genetic structure of the eastern oyster Crassostrea virginica was examined along the coast of North Carolina (NC); utilizing a fragment of the mitochondrial large (16S) ribosomal subunit gene sequence known to distinguish three regional haplotypes in C. virginica—North Atlantic, South Atlantic, and Gulf of Mexico. Significant geographic genetic structure was identified among all populations studied, with little temporal genetic variation observed over the 9-y sampling period. The results are consistent with those of previous studies analyzing geographic variation in mitochondrial DNA16S sequence data, which revealed a genetic discontinuity between North and South Atlantic oyster assemblages along the NC Coast. The increased geographic resolution and duration of sampling in this study revealed a sustained significant shift in genetic composition between Pamlico Sound populations and southern NC populations of C. virginica. These results suggest that a prolonged or persistent barrier impacting larval dispersal or post settlement survival exists between these regions limiting gene flow between North and South Atlantic oyster assemblages.

Field evaluation of mortality from hemolymph extraction as a source of DNA, and application to PCR-RFLP identification of threatened freshwater mussel species.

ABSTRACT Evolutionary convergence and plasticity of shell characters creates great confusion in freshwater mussel systematics and complicates field census efforts. Genetic identification offers a powerful alternative. But methods involving DNA sequencing are expensive and require tissue sampling, whose effects on survival and health of animals from natural populations have seldom been assessed. We used hemolymph sampling as a nonlethal source of tissue for DNA extraction, and developed genetic identification methods using Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis as an alternative for field surveys. We focused on two morphologically similar endemic species in Lake Waccamaw, North Carolina: the abundant Elliptio waccamawensis and the less common Lampsilis fullerkati. These served as models for surveyors who are often faced with conducting accurate census of cryptic rare species that co-occur with more common forms. Hemolymph-sampled and control individuals of these two species and Leptodea ochracea were caged together in enclosures in Lake Waccamaw. Eight-week survival was 100% and we detected no significant effect of hemolymph extraction on shell growth. PCR-RFLP analysis of the 16s rRNA gene reliably identified species and detected cases of morphological misidentification, both in these collections and from belt transects in the lake. We also developed PCR-RFLP markers that distinguished pairs of cryptic taxa from surveys of streams in southeastern North Carolina. Our results show how nonlethal tissue sampling and PCR-RFLP assays designed for a regional fauna can be useful tools in freshwater mussel conservation programs.

Phylogenetic Analysis of Lake Waccamaw Endemic Freshwater Mussel Species

Abstract: About 70% of freshwater mussel species in North America are threatened, endangered, or recently extinct. A large number of the surviving species are endemics. One hotspot for endemic diversity is the southeastern U.S.A. Lake Waccamaw in southeastern North Carolina contains two putative endemic unionids, Lampsilis fullerkati (Johnson, 1984) and Elliptio waccamawensis (Lea, 1863). We used multiple phylogenetic analyses to assess phylogenetic affinities and test the status of these named species. Mitochondrial 16S rRNA gene, cox1, and nad1 sequences were obtained from 109 individuals of the putative endemics and potentially related species from Lake Waccamaw, the Waccamaw River, and the Yadkin/Pee Dee, Little Pee Dee, and Lumber Rivers in the Pee Dee Drainage. In addition to the analysis of each individual gene region, a total evidence analysis, in which all three regions were combined, was conducted using Bayesian phylogenetic methods. All three genes were analyzed using both Bayesian and maximum likelihood methods to test the robustness of the phylogeny of the genus Elliptio (Rafinesque, 1819) and both methods produced consistent topologies with minor differences in the Elliptio “lance” clade. Results suggest that the status of the Lake Waccamaw putative endemics may need to be reconsidered. Lampsilis fullerkati is not phylogenetically distinct from L. radiata (Gmelin, 1791) from outside the lake, and Elliptio waccamawensis groups with and is not genetically distinguishable from E. congaraea (Lea, 1831) individuals from the Waccamaw River.