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Monitoring oyster (Crassostrea virginica) populations in southeast Florida

A research proposal: 2004-2007

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Background: The American oyster (Crassostrea virginica) occupies estuarine and nearshore habitats throughout the eastern and Gulf of Mexico coasts of the United States.  This animal supported a subsistence fishery even before European colonization of the United States (MacKenzie, 1997), and throughout recent history has provided an important economic and cultural resource to coastal inhabitants.  In addition to its direct economic benefits, the oyster also provides essential habitat for many other estuarine inhabitants (Bahr and Lanier, 1981).  The American oyster is one of the most culturally, economically, and ecologically important residents of U.S. coastal waters.

                In Florida, oysters occur along both the Atlantic and Gulf of Mexico coasts in almost all estuarine and nearshore waters.  Along the Atlantic coast, oysters are generally confined within bays and lagoons such as Lake Worth Lagoon or the Indian River.  Those waters, and other coastal waters on the southeast coast of the state, have experienced altered patterns of water delivery and quality as a result of water management practices related to the St. John’s River and Kissimmee River basins, Lake Okeechobee, and the Everglades.  In particular, the redirection of freshwater out of those inland basins and into the aforementioned coastal waters has altered both the timing and the range of salinity variation in those coastal waters.  Alterations in freshwater flow have reduced or eliminated many oyster reef areas and have impacted both the timing and extent of oyster reproduction (Berrigan et al., 1991).  The diverse community associated with the oyster reefs has been impacted to an equivalent or greater degree.

                The Comprehensive Everglades Restoration Program (CERP) is being implemented as a means of restoring, to the greatest degree possible, natural freshwater flow to coastal waters on both coasts of south Florida.  The Monitoring and Assessment Program (MAP) component of CERP is designed to provide a diverse approach to documenting and describing the impacts of changed freshwater flow to the flora and fauna inhabiting inland landscapes and coastal waters.  Because of their wide distribution, historical context, and essential habitat value, the American Oyster is included as a target species for monitoring.  Changes in oyster distribution and abundance will be monitored at a variety of sites on both the Atlantic and Gulf of Mexico coasts of south Florida, including the St. Lucie estuary, the Loxahatchee River, Lake Worth Lagoon, Biscayne Bay, the Ten Thousand Islands area, and the Caloosahatchee River.  Dr. Aswani Volety with Florida Gulf Coast University is leading the effort to monitor oyster populations in the Ten Thousand Islands area and the Caloosahatchee River.  The Molluscan Fisheries research group at the Florida Fish and Wildlife Conservation Commission’s (FWC) Fish and Wildlife Research Institute (FWRI) outlines a proposal for a complimentary monitoring program that will be implemented in the St. Lucie estuary, the Loxahatchee River, Lake Worth Lagoon, and Biscayne Bay.  To provide a background for comparison, outlier oyster populations in Tampa Bay, Mosquito Lagoon, and the Sebastian River will also be monitored.

Methods: Monitoring the status of oyster populations in southeast Florida requires the collection of data concerning the distribution and abundance of adult oysters, the reproductive and health status of those oysters, the rate of recruitment of new oysters to the population, and the growth and survival of young oysters until they join the adult-size class.  These data will be collected in a manner that is complimentary to the data collection methods being employed by Dr. Aswani Volety at the Caloosahatchee River on the Gulf of Mexico coast of Florida.  This complimentary approach ensures, to the greatest degree possible, that data gathered from these concurring projects will be statistically and conceptually comparable.

Adult sampling: We anticipate sampling oyster reefs at seven sites in Florida, four of which are included in the Northern Estuaries component of the CERP MAP and three of which will be outliers that are needed for comparisons of long-term trends not related to CERP activities (Underwood and Chapman, 2003).  The former sites include Biscayne Bay, Lake Worth Lagoon, Loxahatchee River, and St. Lucie estuary, and the latter sites include Sebastian River, Mosquito Lagoon, and Tampa Bay (Pinellas Point).

                On each oyster reef that is sampled within a study site, we intend to haphazardly deploy 1-m²  (one metered squared) quadrats and to harvest all oysters within each quadrat determine the number of live and dead oysters and the shell height (SH = maximum linear distance from umbo to ventral shell margin) of those oysters.  During the first year of the study, adult sampling will be conducted twice. One sampling is scheduled for June, the second in December, to be compatible with the sample schedule for the Caloosahatchee monitoring program.  However, the timing of sampling may be adjusted following the first sample year, dependent upon the timing of recruitment and the temporal patterns of mortality.

                During the initial year of the study, we will sample three oyster reefs (stations) at each study site (Tampa Bay, Mosquito Lagoon, Sebastian River, St. Lucie estuary [three sites], Loxahatchee River [two sites], Lake Worth Lagoon, and Biscayne Bay).  In the event that we cannot find three distinct oyster reefs, for example, in Biscayne Bay, then we will sample all available oyster reefs.  Within the St. Lucie estuary, we will consider the north fork, the south fork, and the middle estuary to be separate sites, and we will sample three randomly selected oyster reefs within each of those sites.  Previous mapping of oyster reefs in the St. Lucie estuary indicates that at least eight reefs exist within each of the north fork, south fork, and middle estuary (Ibis Environmental Inc., 2004), so we anticipate no problems with accessing the required number of reefs.  Within the Loxahatchee River, we will consider the south fork and the northwest fork to be separate sites, and we will sample three randomly selected oyster reefs within each of those sites.  Previous mapping of oyster reefs in the Loxahatchee River indicates that at least 10 oyster reefs are present within each of the south fork and northwest fork (Loxahatchee River District, unpublished data), so we anticipate no problems with accessing the required number of reefs.  For each of the St. Lucie and Loxahatchee systems, this sampling strategy will allow us to separately assess changes in oyster reefs in areas that may be differentially affected by changes in water management practices.

                At each station within a site, we will collect 10 replicate 1-m² quadrat samples of live/dead adult oysters.  That sampling intensity will allow us to detect a true change in the density of oysters that is 1.5 times the standard deviation of oyster density at that site, with a 95 percent probability of detecting a true change when that change occurs (equivalent to a five percent likelihood of a Type I error that we claim a true change when one has not actually occurred).  Of equivalent or greater importance (Underwood and Chapman, 2003), our probability of making a Type II error (that we claim no change in density when one has actually occurred) will be less than 20 percent for a change that is 1.5 times the standard deviation and will be less than five percent for a change that is twice the standard deviation (Table 1).  Thus, if a substantial change in oyster density occurs at any of these sites, ostensibly in response to CERP water management activities, we will have a very high likelihood of detecting that change.

                Recent surveys of oyster reefs were conducted in both the St. Lucie estuary and the Loxahatchee River (URS Greiner Woodward Clyde, 1997; Ibis Environmental Inc., 2004; Loxahatchee River District, unpublished data), but the available data regarding oyster density on each sampled reef was semi-quantitative.  We therefore have no quantitative information on the variance that we can expect in oyster density among sampling stations.  We therefore intend to use the adult density data that we collect during the first year of our study to provide quantitative estimates of sample variance.  We will then use that sample variance data to reevaluate the adequacy of our replication scheme and, if necessary, modify the replication scheme accordingly.  In that regard, we have used relatively conservative ranges of detectable difference (1.5 -and 2-times the standard deviation) for our initial sample size analysis.  We anticipate that the power of our tests will actually be greater than predicted based upon that range of detectable differences.

Table 1: Power as a function of the number of groups (number of stations within a site, with a site being e.g., Lake Worth Lagoon and a station being an oyster reef within that site) and the sample size (number of replicate samples) at each station.  Alpha represents the likelihood of making a Type I error and Power (= Beta) represents the likelihood of making a Type II error.  See text for a description of Type I and Type II error.

Spat Recruitment: We will monitor oyster recruitment at each of the reefs where we conduct adult sampling.  We will use axenic, or sterile, adult oyster shells collected from relict oyster reefs or from other appropriate sources.  Three replicate spat (recently settled oysters) monitoring arrays (arrangements) will be placed at each station along the edge of the reef facing open water and within 2-3 meters of the reef depending upon local conditions and security.  Each recruitment collector, or trap, will consist of 12 oyster shells (5.5 – 7.5 cm SH) strung together with a weighted galvanized wire.  Shells will be placed with their inner surface facing down when suspended off the bottom, and oyster recruitment will be estimated by counting the number of spat on the underside of the strung shells.  Each monitoring collector will be deployed and recovered on a monthly schedule for the entire first year of the study.  If the first year of data indicates that recruitment is limited to certain months of the year, we may limit our deployments to those months during subsequent study years.  For example, Dr. Volety limits his recruit monitoring efforts to the months of March through October because that is the time of peak recruitment in southwest Florida.  However, we don’t have enough information on the timing of oyster recruitment in southeast Florida to support the implementation of a time-limited monitoring program.

Reproductive and Disease Monitoring: We will sample for analysis of gonadal condition, and for the prevalence and intensity of the oyster diseases Perkinsus marinus (“dermo”) and Haplosporidium nelsoni (MSX), on a monthly basis.  A sample of five oysters from each of the three reefs that we monitor at each site (total N = five oysters x three reefs x 10 sites = 150 per month) will be transported live and chilled to the FWRI laboratory for processing.  Each individual will be measured (SH, mm), shucked, and the tissues processed for reproductive and disease condition according to the methods described below.

For reproductive analysis, the gonad will be isolated and fixed in a five percent buffered formalin seawater (isosmotic) solution.  Following 20 hours of fixation, the gonad will be thoroughly rinsed in tap water and preserved in 70 percent ethanol for subsequent histological preparation.  Histological preparation will consist of dehydrating each gonad in 95 percent ethanol for a minimum of three hours, then embedding the gonad in JB4 glycomethacrylate resin, beginning with an exposure to 50 percent JB4 dissolved in 95 percent ethanol followed by two sequential exposures to 100 percent JB4.  At least two 3.5-micrometer sections will be cut from each embedded sample using a microtome mounted with a glass knife, maintaining a minimum separation of 60 micrometers (the approximate maximum diameter of an oocyte) between sections.  Thin sections will be stained with hematoxylin and eosin, then mounted on pre-labeled glass slides for analysis.  Resultant slides will be examined at a total magnification of 200-400x on a compound microscope and each sample assigned to a reproductive stage following a classification scheme (Table 2) modified from the work of Loosanoff (1937), Jaramillio et al. (1993), and Walker and Heffernan (1994).   Qualitative reproductive data will be plotted and the patterns of gonad development and spawning compared among sites.

Table 2.  Qualitative reproductive staging criteria for oysters, Crassostrea virginica, collected from Florida waters.

No Data                                 Unable to read slide

Inactive                                  Gonadal tissue undifferentiated

Developing                           Follicle wall expanded and lumen contains spermatocytes or oocytes

Ripe                                       Follicular areas filled with either dense or radiating bands of sperm or ova

Early Spawning                    Central lumen of either male or female follicles beginning to empty

Spawning                              Lumen of follicles more extensively emptied

Spent                                     Follicle lumen emptied of ova and spermatids, many amoebocytes and cellular debris

                                                often present

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                The tissue that remains following the extraction of the gonad will be utilized for assessment of disease condition.  Dermo prevalence and intensity will be diagnosed using Ray’s fluid thyoclycollate method (RFTM), as described by Bushek et al. (1994).  Small (1 cm²) pieces of gill and mantle tissue will be incubated in RFTM with antibiotics for four days at 25^o C (77^o F). Tissue pieces will then be placed on glass microscope slides, macerated, or softened, with a razor blade, stained with Lugol’s, and examined at a total magnification of 40-times for the presence of hypnospores.  Parasite density (infection intensity) will be ranked using the Mackin scale, which ranges from zero (no infection) to five (heavy infection).  Average parasite densities will be calculated for each sample.  Remaining oyster tissue will be placed in Dietrich’s fixative and processed for histopathology (Barber, 1996).  Finished slides will be examined at a total magnification of 100-times for the presence of MSX parasites.  Infection will be classified as either light (epithelial) or heavy (systemic).  Mean prevalence will be calculated for each site.  Infection prevalence and intensity will be statistically compared among sample dates and locations with the non-parametric Friedman’s Test (Zar, 1974).

                The data derived from this sampling regime will allow us to resolve reproductive and disease patterns at two spatial scales: within the reef at each site and among sites.  Understanding variation within each site will be necessary in order to properly analyze differences among sites or among dates within each site.  The resultant data also will be useful for comparison with a similar monitoring program being conducted by Dr. Volety in southwest Florida (Volety et al., 2003).  Finally, the disease data will be useful for comparison with data on MSX and Dermo distribution and intensity studies conducted throughout the Atlantic and Gulf of Mexico coasts of the United States.

Juvenile Growth Monitoring: We will plant juvenile oysters at all study stations at all study sites to monitor growth and mortality.  The juvenile oysters that are planted at each site will be cultured from parent broodstock, or young, collected at that site or, in the event that adult oysters are not available at that site (e.g., Biscayne Bay), at the next nearest study site from which adult oysters can be obtained.

                Adult oysters will be collected from each of the six east coast and one west coast study sites during February or March of each year.  Oysters collected from each of the east coast sites will be separately maintained and transported to the spawning facility, tentatively Harbor Branch Oceanographic Institution in Fort Pierce.  The exact number of broodstock oysters to be provided from each site will be determined by Dr. John Scarpa of Harbor Branch, but we anticipate providing approximately 10-20 oysters from each site.  The broodstock from each site will be separately maintained and spawned at the Harbor Branch facilities, and the resultant offspring also will be separately maintained.  Offspring from each site will be raised to a shell height of 10-20 mm then transported to their respective field sites for planting.  Spawning will occur in February or March of each year and field planting will occur approximately three months later depending upon growth rate in the hatchery.

                We will plant 400 juvenile oysters at each station at each site.  Thus, we will need to plant 1200 oysters at each of the Mosquito Lagoon, Sebastian River, Lake Worth Lagoon, and Biscayne Bay sites.  At the St. Lucie estuary site, we will plant 400 oysters at each of the three stations in each of the north fork, south fork, and middle estuary of that system, for a total planting of 3,600 oysters.  In the Loxahatchee River, we will plant 400 oysters at each station in each of the northwest and south forks of the river, for a total planting of 2,400 oysters.

                Oysters will be planted into 0.5 mm mesh cages at all stations.  Each cage will be composed of two compartments, with one compartment completely enclosed by mesh to exclude all predators larger than 0.5 mm and the other compartment partially open to allow entry of such predators.  This approach will allow us to assess relative mortality at each site that can be attributed to macrofaunal predators.

                Cages will be samples on a monthly schedule.  The number of live oysters in each of the two compartments in each cage will be determined, and the SH of a haphazard sample of 30 oysters (or all remaining live oysters if < 30) will be measured to the nearest 0.1 mm using Vernier calipers.  Oysters from each year and class will be monitored until they reach a mean asymptotic size or for as long as oysters remain alive in the cage (whichever comes first), but a new brood of oysters will be planted and monitored each year.

                An identical approach will be used for assessment of juvenile oyster growth at the Tampa Bay study site, with the exception that broodstock collected from the Tampa Bay reef will be spawned and the offspring raised at a hatchery that is located on the Gulf of Mexico coast.  This oyster spawning operation will be conducted in cooperation with Dr. Volety who will be following a similar protocol for his southwest Florida research.  Conducting all east coast spawning activities on the east coast and all west coast spawning activities on the west coast will eliminate the possibility of genetic or disease cross-contamination between coasts.

Water Quality Monitoring: Monthly water quality sampling will be conducted in conjunction with field sampling at each study site.  Parameters to be monitored will include water depth, temperature, salinity, conductivity, pH, dissolved oxygen, and turbidity.  Water depth will be determined with sounding line and turbidity will be obtained using a standard Secchi disk.  For temperature, salinity, conductivity, pH, and dissolved oxygen, a water column profile will be obtained using a multi-parameter data logger.  The data logger will be calibrated according to manufacturer’s specifications, and calibration logs will be provided with each quarterly report.  Ancillary data collected during each sampling episode will include time of day, weather conditions, tide stage, and any unique observations that may be pertinent to the study.