John Supan

High resolution mapping and classification of oyster habitats in nearshore louisiana using sidescan sonar

Sidescan sonar holds great promise as a tool to quantitatively depict the distribution and extent of benthic habitats in Louisiana’s turbid estuaries. In this study, we describe an effective protocol for acoustic sampling in this environment. We also compared three methods of classification in detail: mean-based thresholding, supervised, and unsupervised techniques to classify sidescan imagery into categories of mud and shell. Classification results were compared to ground truth results using quadrat and dredge sampling. Supervised classification gave the best overall result (kappa=75%) when compared to quadrat results. Classification accuracy was less robust when compared to all dredge samples (kappa=21–56%), but increased greatly (90–100%) when only dredge samples taken from acoustically homogeneous areas were considered. Sidescan sonar when combined with ground truth sampling at an appropriate scale can be effectively used to establish an accurate substrate base map for both research applications and shellfish management. The sidescan imagery presented here also provides, for the first time, a detailed presentation of oyster habitat patchiness and scale in a productive oyster growing area.

Effectiveness of icing as a postharvest treatment for control of Vibrio vulnificus and Vibrio parahaemolyticus in the eastern oyster (Crassostrea virginica).

The focus of this research was to investigate the efficacy of icing as a postharvest treatment for reduction of the levels of Vibrio vulnificus and Vibrio parahaemolyticus in commercial quantities of shellstock oysters. The experiments were conducted in June and August of 2006 and consisted of the following treatments: (i) on-board icing immediately after harvest; (ii) dockside icing approximately 1 to 2 h prior to shipment; and (iii) no icing (control). Changes in the levels of pathogenic Vibrio spp. during wholesale and retail handling for 2 weeks postharvest were also monitored. On-board icing achieved temperature reductions in all sacks in accordance with the National Shellfish Sanitation Program standard, but dockside icing did not meet this standard. Based on one-way analysis of variance, the only statistically significant relationship between Vibrio levels and treatment occurred for samples harvested in August; in this case, the levels of V. vulnificus in the noniced oysters were significantly higher (P < 0.05) than were the levels in the samples iced on-board. When analyzing counts over the 14-day storage period, using factorial analysis, there were statistically significant differences in V. vulnificus and V. parahaemolyticus levels by sample date and/or treatment (P < 0.05), but these relationships were not consistent. Treated (iced) oysters had significantly higher gaping (approximately 20%) after 1 week in cold storage than did noniced oysters (approximately 10%) and gaping increased significantly by day 14 of commercial storage. On-board and dockside icing did not predictably reduce the levels of V. vulnificus or V. parahaemolyticus in oysters, and icing negatively impacted oyster survival during subsequent cold storage.

NONLETHAL SPERM COLLECTION AND CRYOPRESERVATION IN THE EASTERN OYSTER CRASSOSTREA VIRGINICA

ABSTRACT 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 ± 1.0 × 103 cells/mL (in 2 L seawater). No oysters (n = 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 4.5 × 105 to 2.3 × 108 cells per male. Mechanical notching did not cause mortality to oysters (n = 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 × 107 cells by 18-G needle (n = 8) with fresh motility of 16 ± 12% and postthaw motility of 3 ± 2%, and 1.35 × 108 cells by 20-G needle (n = 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 × 108 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 = 0.550), postequilibration motility (P = 1.000), postthaw motility (P = 0.101), fresh membrane integrity (P = 1.000), or postthaw membrane integrity (P = 1.000), but a difference was observed in fertilization (P = 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.

Consumer Method To Control Salmonella and Listeria Species in Shrimp

The purpose of this study was to determine whether the current consumer method of boiling shrimp until floating and pink in color is adequate for destroying Listeria and Salmonella. Shrimp samples were submerged in bacterial suspensions of Listeria and Salmonella for 30 min and allowed to air dry for 1 h under a biosafety cabinet. Color parameters were then measured with a spectrophotometer programmed with the CIELAB system. Twenty-four shrimp samples were divided into groups (days 0, 1, or 2) and stored at 4°C. The samples were treated by placing them in boiling water (100°C) on days 0, 1, and 2. The shrimp were immediately removed from the boiling water once they floated to the surface, and color parameters were measured. Bacterial counts were determined, and the log CFU per gram was calculated. The effect of sodium tripolyphosphate on the color change of cooked shrimp also was determined. Initial bacterial counts on shrimp after air drying were 5.31 ± 0.14 log CFU/g for Salmonella Enteritidis, 5.24 ± 0.31 log CFU/g for Salmonella Infantis, 5.40 ± 0.16 log CFU/g for Salmonella Typhimurium, 3.91 + 0.11 log CFU/g for Listeria innocua, 4.45 ± 0.11 log CFU/g for Listeria monocytogenes (1/2a), and 3.70 ± 0.22 log CFU/g for Listeria welshimeri. On days 0, 1, and 2, all bacterial counts were reduced to nondetectable levels for shrimp samples that floated. The average time for shrimp to float was 96 ± 8 s. The bacterial counts remained at nondetectable levels (<10 log CFU/g) during refrigerated (4°C) storage of cooked shrimp for 2 days. The redness, yellowness, and lightness were significantly higher (P < 0.0001) for the cooked shrimp than for the uncooked shrimp on all days tested. The standard deviation for redness in the cooked shrimp was large, indicating a wide range of pink coloration on all days tested. The results suggest that boiling shrimp until they float will significantly reduce Listeria and Salmonella contamination, but color change is not a good indication of reduction of these pathogens because of the wide natural color variation.

Rapid Estimation of Gonad-to-Body Ratio in Eastern Oysters by Image Analysis

Abstract The goal of this study was to develop a rapid and reliable method of quantifying the gonadal condition of eastern oysters Crassostrea virginica based on a gonad-to-body ratio (GBR). The objectives were to compare a previously established transect method with three alternative computer-based image analysis (IA) methods based on acquisition time, composite GBR values, and GBR values at different stages of gonadal development. The first IA method calculated the area of the gonad and body while eliminating the dorsal and ventral curvature of the gonad, the second calculated the area of the gonad and body while eliminating the gill area, and the third calculated the total area of the gonad and body. The GBR values from the first and third IA methods were not different from those from the transect method. The second IA method resulted in higher GBR values in 80% of the measurements compared with the other three methods. However, measuring the gonadal area and total body area was five times faster than ...

Production of Calcium-Binding Proteins in Crassostrea virginica in Response to Increased Environmental CO2 Concentration

Biomineralization is the complex process by organisms produce protective and supportive structures. Employed by mollusks, biomineralization enables creation of external shells for protection against environmental stressors. The shell deposition mechanism is initiated in the early stages of development and is dependent upon the concentration and availability of calcium carbonate ions. Changes in concentrations of the critical ions required for shell formation can result in malformation of shells. As pCO2 concentrations in the atmosphere continue to increase, the oceans are becoming more acidified. This process, known as ocean acidification (OA), has demonstrated adverse effects on shell formation in calcifying organisms across taxa. Although OA is known to inhibit the shell deposition in mollusks, the impact of OA on the gene regulation of calcium deposition remains unknown. Here we show the responses of four calcium-binding protein genes, caltractin (cetn), calmodulin (calm), calreticulin (calr), and calnexin (canx), to CO2-derived OA using a Crassostrea virginica mantle cell (CvMC) culture model and a larval C. virginica model. These four genes were cloned from C. virginica and the three-dimensional structures of the proteins encoded by these four genes were fully characterized using homologue modeling methods. Although an acidified environment by increased atmospheric pCO2 (1000 ppm) did not result in significant effects on CvMC proliferation and apoptosis, lower environmental pH induced upregulations of all four calcium-binding protein genes in CvMCs. Similarly, increased pCO2 did not affect the growth of larval C. virginica in the early stages of development. However, elevated pCO2 concentrations enhanced the expression of these calcium-binding protein genes at the protein level. The four calcium-binding protein genes demonstrated responsive expression profiles to an acidified environment at both cellular and individual levels. Further investigation of these genes may provide insight into the molecular regulation of mollusk biomineralization under OA stress.