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Intrapopulational variation in Vibrio vulnificus levels in Crassostrea virginica (Gmelin 1971) is associated with the host size but not with disease status or developmental stability.

ABSTRACT Vibrio vulnificus is an important human pathogen, which occurs naturally in estuarine waters and in large numbers in oysters. The levels of V. vulnificus in shellfish vary greatly, and the causes of this variation are not well understood. A protozoan parasite, Perkinsus marinus, is a pathogen of oysters, which was earlier suggested to increase susceptibility of oysters to bacterial infections. A goal of our study is to determine whether the presence of P. marinus plays a role in determining the incidence or magnitude of V. vulnificus infection in oysters. We also used fluctuating asymmetry (FA) of shell weight as an indicator of developmental instability and thus of microbially-induced physiological stress in oysters. We harvested 155 adult oysters (Crassostrea virginica) from a small environmentally homogeneous site in North Carolina, and examined them for V. vulnificus and P. marinus infections. V. vulnificus and P. marinus were detected in 88% and 73% of the oysters, respectively. The incidence of the two pathogens in these oysters was independent, with no greater tendency to infection by V. vulnificus in oysters infected by P. marinus and vice versa. V. vulnificus loads per g tissue weight varied by over four orders of magnitude in oysters of the same size, weight and age, which were harvested from this single habitat. V. vulnificus loads were found to be unaffected by age of oysters (and thus by the length of exposure to this pathogen). On the contrary, there was a significant decrease in both incidence of V. vulnificus infection and tissue loads of this bacterium with the host weight, possibly indicating growth dilution. Infection with V. vulnificus or P. marinus did not affect FA of shell weight in oysters. Overall, our findings suggest that infection by P. marinus does not predict V. vulnificus loads in C. virginica, nor did oysters infected with either of these pathogens exhibit physiological stress that results in developmental instability.

KEY WORDS: Vibrio vulnificus, Perkinsus marinus, fluctuating asymmetry, Crassostrea virginica, intrapopulational variability


Vibrio vulnificus is an important human pathogen common in estuarine and coastal waters around the world, and oysters serve as major vectors in the transfer of this bacterium to humans. V. vulnificus infection is a serious concern for the oyster industry and public health in the United States, particularly in Gulf of Mexico states. Ingestion of raw or undercooked seafood, especially oysters, containing this bacterium can result in illness and even death (0liver & Kaper 2001). Controlled purification of shellfish (e.g., depuration) and other postharvest treatments have been extensively used worldwide in commercial operations to remove unwanted microorganisms from shellfish and thus to improve product quality. However, despite years of study, there are no known methods to produce fresh, raw oysters containing levels of V. vulnificus at "safe" levels (3 MPN [g.sup.-1] or less). Unlike fecal coliforms, V. vulnificus is not removed from oyster tissues by controlled purification methods (Tamplin & Capers 1992); in fact the number of V. vulnificus cells may significantly increase in live shellfish subjected to control depuration (Harris-Young et al. 1995). Moreover, this bacterium has been reported to persist in oysters at high levels and even reproduce in oyster hemolymph and other tissues (Tamplin & Capers 1992).

Dermo disease caused by an intracellular protozoan parasite, Perkinsus marinus, has a high occurrence in wild and cultured oyster (Crassostrea virginica, Gmelin 1971) populations and is a likely candidate to create physiological conditions favoring bacterial infections including those by V. vulnificus. P. marinus infection greatly weakens oysters, resulting in increased morbidity and mortality, and laboratory studies with isolated oyster hemocytes suggest that these protozoan parasites may lead to impaired ability of oysters to resist bacterial infection. Thus, Tall et al. (1999) demonstrated that P. marinus produces a serine protease which suppresses the vibriocidal activity of hemocytes in infected oysters. Similarly, La Peyre and Volety (1999) showed that extracellular proteins produced by P. marinus inhibit the capacity of hemocytes to kill V. parahaemolyticus. Similar results were reported for other bivalves infected by Perkinsus spp. supporting findings in C. virginica and indicating that Perkinsus-induced inhibition of host bactericidal defense is a common phenomenon. Thus, phagocytic rates and antibacterial activity of hemocytes was significantly suppressed in clams infected with P. atlanticus (Ordas et al. 2000) and by exposure of isolated hemocytes to secretion products released by cultured P. atlanticus (Ordas et al., 1999). Such studies clearly indicate that the presence of P. marinus in oysters may result in their reduced resistance to bacterial infections. However, the relationship between protozoan infections and Vibrio loads in whole oysters has not been reported. Therefore, one aim of this study is to test the hypothesis that oysters infected with P. marinus carry heavier loads of V. vulnificus than uninfected individuals from the same populations.

Physiological stress resulting from infection with parasites or pathogens is often reflected in an impairment of developmental stability, which is defined as the ability of an organism to produce a consistent phenotype in a specific environment (Zakharov 1989, Parsons 1990). Increase in developmental instability (DI) due to variety of environmental and intrinsic stressors, including parasitic and bacterial diseases, has been reported in various animal populations (review in: Polak 1997, Moller & Swaddle 1997, Hoffmann & Woods 2003), where it is most often measured by fluctuating asymmetry (FA), the random variation in the difference between right and left sides of bilateral traits (Palmer & Strobeck 1986). Thus, an increase in FA in infected individuals could be interpreted as a marker of physiological stress experienced by an organism due to parasites or pathogens. However, it is not known whether parasitic infection by either P. marinus or V. vulnificus would increase FA levels and thus developmental instability in oysters.

The purpose of this study is to evaluate the natural levels of variability in V. vulnificus loads in a wild oyster population and to test whether the bacterial loads increase with increasing oyster age (and thus the length of exposure to the pathogen), weight or shell length, or with P. marinus infection. We also tested whether P. marinus or V. vulnificus infection leads to an elevated level of developmental instability in an oyster population, which would indicate parasite-induced physiological stress.


Animal Collection and Maintenance

Oysters were collected on June 15, 2004 from a subtidal habitat in Stump Sound, North Carolina. One hundred and fifty-five adult oysters (55-155 mm valve length) were randomly collected from a small area (ca. 100 x 100 m) of homogenous soft-bottom habitat to ensure that all organisms used in the present study were exposed to the same environmental conditions. Age of oysters within this size range was 2-5 y as determined by the count of annual growth checks on their shells. Age determination by growth checks was verified by comparison to the growth checks on cultured oysters of known age grown subtidally in the nearby area of Stump Sound (courtesy of J. Swartzenberg, J & B Aquafood). Water temperature at the time of collection was 26[degrees]C and salinity was 31 [per thousand]. Oysters were immediately placed on ice and transported to the University of North Carolina at Charlotte within 5 h of collection for further processing and analysis. Processing of oyster tissues was completed within 24 h of collection. During this time, oysters were kept on ice to prevent postharvest build-up of bacteria.

Determination of V. vulnificus Loads and P. marinus Infection

Oysters were externally cleaned and opened with an alcohol-flamed oyster knife. The oyster contents were removed, weighed, and homogenized in sterile blender jars with an equal volume of sterile diluent (50% artificial sea water). Homogenates were diluted and plated for V. vulnificus, using the cellobiose-polymyxin B-colistin (CPC) agar developed in our laboratory (Massad & Oliver 1987) and which has been used by us and others for the primary isolation of V. vulnificus (Harwood et al. 2004, Oliver 2003). When colonies of appropriate color and morphology are selected, this medium has been shown to be 82% accurate in the isolation of V. vulnificus (Sun & Oliver 1995b). Using these same criteria, Sloan et al. (1992) found 81% of the typical V. vulnificus colonies on CPC to be identified as this species.

Small samples of gill tissue (50-100 mg) were removed prior to homogenization and placed in DNA fixing solution for DNA extraction and for PCR diagnostics of P. marinus. Remaining tissues were weighed to the nearest 0.01 g, and the maximum valve length was measured to the nearest 0.1 mm. A total of 155 oysters were available for the analysis. For diagnostics of P. marinus, total DNA was isolated from 50-100 mg samples of gill tissue following an improved protocol for DNA isolation from mollusks developed by Sokolov (2000). This method allowed us to isolate total DNA, which in infected oysters contained DNA of P. marinus in addition to the host (oyster) DNA. Determination of P. marinus infection was performed using PCR with the following primers:



These primers are highly specific for P. marinus (Penna et al. 2001), and do not cross-amplify with oyster DNA or other parasites. Optimized PCR conditions for P. marinus detection were as follows: 25 [micro]L of reaction volume containing 1x PCR buffer, 2 mM Mg[Cl.sub.2], 100 [micro]M of dNTPs, 0.7 U of Taq polymerase, 150 ng of each P. marinus primer and 50-100 ng of template DNA is subjected to one denaturation cycle at 94[degrees]C for 5 min, 35 cycles at 94[degrees]C for 45 s, 55[degrees]C for 30 s and 72[degrees]C for 45 s and one final extension cycle at 72[degrees]C for 7 min P. marinus DNA obtained from monocultures of this parasite (gift of Dr. G. Vasta) was used as a positive control. Amplified DNA fragments were resolved on ethidium bromide-stained 1.5% agarose gels and screened for the presence of a ca, 304 bp product characteristic of P. marinus, which indicated infection of the oyster with this parasite. This method is highly sensitive with detection limits of 0.1 pg DNA of P. marinus, corresponding to 1 protozoan cell [g.sup.-1] oyster tissue (Penna et al. 2001).

Fluctuating Asymmetry Measure

To obtain a measure of fluctuating asymmetry for these oysters, we weighed both left and right valves (to the nearest mg) in all individuals whose valves were not broken (total = 120) as described in Frechette et al. (2003). After one round of weighing, another entire round was done to ensure that two weights for both the left and right sides of each oyster were available. This allowed an estimation of the extent of measurement error via a 2-way ANOVA in which the two factors were sides and individuals (Palmer & Strobeck 1986). In this model, the factor "Individuals" assesses the variation in valve weight among all oysters, and the factor "Sides" assesses whether one side is consistently heavier than the other (directional asymmetry = DA); the individuals by sides interaction assesses FA, and the error assesses replication variation or measurement error (Palmer & Strobeck 1986). Results of this ANOVA showed statistical significance (P < 0.01) for individuals, sides and their interaction, with negligible measurement error (contribution to the total variation = 0.003%). The individuals by sides interaction (FA) component contributed 14.4% of the total, whereas differences among individuals contributed the greatest amount (85.6%) to the total variation.

After this preliminary assessment of DA, FA and measurement error, it was necessary to calculate a measure of FA of valve weight in each oyster for the analysis. The first step in this process typically is to examine the distribution of the differences between right and left sides (R-L) to see whether any DA (directional asymmetry) or antisymmetry is present (Palmer & Strobeck 2003). As explained earlier, DA occurs when one side is consistently larger than the other (as in the mammalian heart). Antisymmetry is usually seen as a bimodal distribution of right minus left differences. For example, claw sizes in male fiddler crabs show an antisymmetric distribution because typically the right claw is enlarged in half the males and the left claw is enlarged in the other half. We therefore first calculated signed differences of weights of the right and left valves in each oyster, and found that the mean of these differences was significantly different from zero, suggesting that DA was present. The distribution of these signed differences was found to be normal, however, suggesting that antisymmetry was not present (Palmer & Strobeck 1986).

Although several measures of FA are in use, we chose the most widely used measure, the absolute or unsigned differences of right minus left sides ([absolute value of R-L]). We therefore calculated these unsigned differences for the valve weights in each oyster but after first subtracting the mean of the right minus left differences from all signed differences between sides to correct for DA. Once these absolute corrected differences were calculated, we tested to see whether they were correlated with the overall size [(L + R)/2] of the oyster valves. Palmer (1994) has shown how scaling effects can sometimes be a problem in asymmetry studies, especially if the magnitude of asymmetry depends on the mean character size itself that varies among samples (in this case, infected vs. noninfected groups). This correlation was significant (P < 0.01) and we therefore logarithmically transformed the left and right side values for each oyster as recommended by Palmer and Strobeck (2003) to eliminate this scaling effect. The final FA measures therefore represented the DA-corrected, absolute values of the logarithmically transformed right minus left side differences of valve weights.

Statistical Analysis

To test for potential associations of the tissue weight, length and age of the oysters with their infection by P. marinus or V. vulnificus, two separate analyses were conducted. The first focused on the incidence of infection by making use of all oysters and coding them each as either 0 or 1, depending on whether they were infected by V. vulnificus or P. marinus. The second analysis used the (log-transformed) actual CFU [g.sup.-1] counts for V. vulnificus among the infected oysters to analyze the magnitude of infection of this pathogen. Prior to both of these analyses, we examined the distribution of length and weight and found length to be normally distributed but weight to be highly skewed (P < 0.01). Weight therefore was log-transformed, and this was successful in achieving normality for this character.

We used the 0/1 values for both P. marinus and V. vulnificus to first test whether there was an association between the incidence of each of these pathogens in the oysters. This was accomplished via a 2 x 2 contingency table analysis in which a phi-coefficient was calculated (Zar 1984). For the 2 x 2 model, this coefficient varies between -1 and 1, and was tested for a significant deviation from zero with the [chi square] statistic generated in the analysis (Zar 1984). A significant phi coefficient would imply that the incidence of the two pathogens is correlated, whereas a nonsignificant result (phi coefficient not different from zero) would suggest that there is no correlation of incidence of the two pathogens in the oysters.

We used logistic regression (Sokal & Rohlf 1995) to test whether weight, length or age (independent variables) of the oysters influenced their incidence of infection (dependent variable) with P. marinus and with V. vulnificus. This procedure yields a test of the overall effect of the three variables on the incidence of infection, as well as separate regressions for each of the three variables with individual tests of their significance. Because two separate analyses were conducted, these effects were evaluated with an adjusted significance level (0.05/2 = 0.025) suggested by the sequential Bonferroni procedure (Rice 1989). Finally, we used multiple regression to test for the effect of weight, length and age on the magnitude of infection of V. vulnificus (log-transformed CFU[g.sup.-1] data).

We tested whether infection with P. marinus or V. vulnificus significantly affected FA levels in valve weight by using the FA measures in separate 1-way analyses of variance (Palmer & Strobeck 2003). In addition, to test for any relationship in FA levels with the magnitude of infection of V. vunificus (log-transformed CFU [g.sup.-1] data), we used simple linear regression of FA on the infection data.


Studied oysters varied from 2 to 5 y in age, the majority (55%) being 3 y of age. Average length of the oysters was 102.8 [+ or -] 1.39 mm, average tissue weight 8.12 [+ or -] 0.13 g (n = 155). Correlations between weight and length (+0.59), weight and age (+0.43) and length and age (+0.42) all were positive in sign and statistically significant (P < 0.01). There was a large variation in V. vulnificus levels in the studied oysters, with individual values ranging from nondetectable levels (<100 CFU [g.sup.-1]) to over [10.sup.6] CFU [g.sup.-1] (Fig. 1).


A total of 42 oysters (27%) were not infected with P. marinus, but only 18 (12%) were not infected with V. vulnificus, as indicated by lack of isolation on CPC agar. Of the 137 infected with V. vulnificus, the mean of the logged CFU [g.sup.-1] counts was nearly 4. The number of oysters that were infected with both pathogens was 98, whereas only 4 were not infected with either pathogen and 52 oysters were infected with either P. marinus or V. vulnificus. A contingency table analysis generated a nonsignificant phi coefficient of -0.04 for the correlation of the incidence of infection of these two pathogens ([chi square] = 0.05, P = 0.83), suggesting that the incidence of these two pathogens is independent. In other words, if an oyster is infected with one of these two pathogens, there is not a greater tendency for it to harbor the other pathogen.

The incidence of infection of neither P. marinus nor V. vulnificus was affected by weight, length and age of the oysters, although overall significance was nearly reached (P = 0.062) for V. vulnificus (Table 1). Inspection of the probabilities of the regressions associated with the individual characters shows that V. vulnificus is most affected by oyster weight and length but not age. If age is omitted and this analysis rerun with just weight and length, it yields an overall probability of 0.026, significant by conventional standards but not quite by the more stringent Bonferroni-corrected standard of 0.025. Thus there is a nearly significant trend for effects of oyster weight and length on the incidence of infection of V. vulnificus (but not of P. marinus). The signs of the regressions on weight and length in the analysis for V. vulnificus indicate that infected oysters tend to be longer but weigh less (mean length and weight = 103.7, 0.902) than uninfected ones (mean length and weight = 95.8, 0.933).

Oyster weight, length, and age and the incidence of P. marinus collectively have a significant effect (P = 0.02) on the magnitude of infection by V. vulnificus, although inspection of the individual regressions shows that this effect is primarily due to weight (Table 2). Thus weight of the oyster is significantly associated with its level of infection, and the negative regression coefficient indicates that oysters that weigh less have a greater magnitude of infection (Table 1, Fig. 2).


One-way analyses of variance showed no significant differences between FA in valve weights between oysters infected and not infected with P. marinus (F = 0.63, DF = 1,118, P = 0.43) or with V. vulnificus (F = 0.07, DF = 1,118, P = 0079). Further, the valve weight FA measures also were not significantly correlated (P = 0.31) with the magnitude of infection of V. vulnificus. In general, therefore, infection of either pathogen does not seem to be affecting developmental instability (as measured by FA in valve weights) in this population of oysters.


Physiological stress caused by P. marinus infection in oysters has been shown to result in host lesions, weight loss (Ray 1954), changes in physiological processes (Paynter 1996) and mass mortalities (Burreson & Ragone Calvo 1996) suggesting that this protozoan pathogen greatly weakens the host and adversely affects host physiology. Studies of oyster immune defense in vitro have also shown a reduction of bactericidal activities of isolated oyster hemocytes by secretory products of P. marinus (Anderson 1999, La Peyre & Volety 1999, Tall et al. 1999). A decrease in hemocyte motility and phagocytosis observed in P. marinus-infected oysters was suggested to favor tissue invasion by bacteria (La Peyre et al. 1995, Garreis et al. 1996). However, the present study using whole oysters does not support this hypothesis and shows that P. marinus infection does not favor development of a common bacterium, V. vulnificus, in C. virginica. Indeed, there were no significant differences in the incidence of infection (% of oysters carrying V. vulnificus), or in the total tissue burden of V. vulnificus (CFU [g.sup.-1] wet weight) between oysters harboring P. marinus and their P. marinus-free counterparts.

Two possible hypotheses can explain this discrepancy between in vitro results and our field studies. Firstly, P. marinus-induced decrease in phagocytic activity of oyster hemocytes may be compensated in vivo by either an increase in the number of active circulating hemocytes, or by humoral immune defense. Indeed, the number of circulating blood cells tends to be elevated in P. marinus-infected oysters (Anderson et al. 1992, Anderson et al. 1995, La Peyre et al. 1995). Lysozyme concentration and hemagglutination titer in P. marinus-infected oysters was similar to (Chu & La Peyre 1989, Chu & La Peyre 1993b, Chu et al. 1993) or higher than (Chu & La Peyre 1993a) in their uninfected counterparts, indicating that the humoral immune defense was not compromised and in some cases even stimulated by P. marinus infection. Interestingly, similar stimulation of the humoral immune defense by P. atlanticus was found in carpet shell clams (Ruditapes decussatus), which showed elevated serum lysozyme and agglutinin levels (Ordas et al. 2000). This suggests that protozoan infection, although resulting in an overall physiological stress in oysters, may not necessarily lead to a decreased bactericidal ability of their immune system due to compensation and/or humoral stimulation mechanisms.

Alternatively, the absence of correlation between P. marinus infection and tissue loads of V. vulnificus in the studied population may be explained if other host factors not related to the innate immunity play a critical role in determination of V. vulnificus infection in oysters. For example, for the surface-associated pool of V. vulnificus, surface properties of oyster gut or mantle epithelia providing attachment sites for bacterial cells may be more important than bactericidal properties of their immune system. On the other hand, innate immunity would play a key role in controlling levels of V. vulnificus pool residing within oyster tissues and in hemolymph. There is evidence that V. vulnificus exists not only in the oyster gut and on body surfaces, but is also present within oyster tissues. Tamplin and Capers (1992) reported that whereas 55% of V. vulnificus cells resided within the digestive tract, 35% were found within the adductor muscle, and less than 10% were found in mantle, gills and hemolymph. A similar finding was reported by Sun and Oliver (1995a), who found 2.7 x [10.sup.5] V. vulnificus cells (>95%) to be present within oyster tissues, whereas an average of 1.3 x [10.sup.4] was found associated with oyster meat surfaces. Interestingly, a recent study has shown that immunosuppression due to either stress-induced or artificial (through experimental injection) elevation of noradrenalin levels in hemolymph resulted in a dramatic increase of V. splendidus loads in the oyster C. gigas (Lacoste et al. 2001a, Lacoste et al. 2001b). This suggests that at least for some Vibrio species, host innate immunity plays a key role in keeping infections at bay. Whether this is the case with V. vulnificus and what is the relative contribution of the host immune system and tissue surface properties to the control of the whole-organism loads of V. vulnificus, is not known. This question requires further investigation.

Interestingly, neither incidence nor loads of V. vulnificus in oysters increased with oyster age indicating that duration of exposure to this bacterium is not a critical factor in determining prevalence and/or magnitude of infections in adult shellfish. On the contrary, V. vulnificus loads measured on a per gram basis significantly decreased with increasing tissue weight of oysters. This may indicate growth dilution in fast growing animals and/or a decrease in surface-volume ratio in large animals leading to a decrease in the relative abundance of surface-associated V. vulnificus pool. However, even in the largest oysters the loads of V. vulnificus ([10.sup.3]-[10.sup.5] CFU [g.sup.-1]) were often above the levels considered safe for the human consumption (3 MPN [g.sup.-1] or less, Oliver & Kaper 2001).

As a corollary, our data suggest that disease status (presence or absence of P. marinus infection) is a poor predictor of V. vulnificus loads in field populations of C. virginica. Fluctuating asymmetry in valve weight did not increase in oysters infected with P. marinus or V. vulnificus, indicating that physiological stress due to infection with these microorganisms did not result in developmental instability of shell weight. Interestingly, there was no increase in V. vulnificus loads with the age of oysters (and thus the length of exposure to this pathogen) but a significant decrease in both incidence of V. vulnificus infection and tissue loads of this bacterium with the host weight, However, large individual variation in tissue loads of V. vulnificus, exceeding four orders of magnitude, was observed in oysters of the same size, weight and age grown in the same habitat, and these may be associated with other host characteristics not analyzed in the present study or individual genetically determined susceptibility to bacterial infection. The latter possibility is currently being studied in our laboratories. Further investigations into the factors controlling this large individual variation of V. vulnificus in oysters are required to understand the ecology and dynamics of this important human pathogen in estuaries and its transfer to humans.


The authors thank Paige Waymer, who assisted with weight determinations; Dr. Thomas Rosche, Ben Smith, Erin Parker and Bryn Adams for their assistance in determining the V. vulnificus levels; Dr. Gerardo Vasta for his gift of P. marinus DNA and Jim Swartzenberg of J & B Aquafood for his help with animal collection. This work was funded in part by North Carolina Sea Grant (RMG-0401), to I.M.S. and JCSU MBRS-RISE Program NIGMS 58042 to M.H. Publication costs were in part covered by the UNC Charlotte.


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I. M. SOKOLOVA, (1) * L. LEAMY, (1) M. HARRISON (2) AND J. D. OLIVER (1)

(1) Biology Department, University of North Carolina at Charlotte, 9201 University City Blvd., Charlotte, North Carolina 28223; (2) Johnson C. Smith University, 100 Beatties Ford Rd., Charlotte, North Carolina 28216

* Corresponding author. E-mail:
Logistic regression of oyster weight, length, and age on the incidence
of infection of P. marinus and V. vulnificus.

 Variable DF Regression Error [chi square] P

P. marinus
 All variables 3 3.788 0.285
 Weight 1 0.882 1.102 0.642 0.423
 Length 1 -0.013 0.013 1.078 0.299
 Age 1 0.485 0.315 2.373 0.124

V. vulnificus
 All variables 3 7.345 0.062
 Weight 1 -3.290 1.641 4.021 0.045 *
 Length 1 0.049 0.021 5.773 0.016 *
 Age 1 0.090 0.401 0.050 0.823

* = P < 0.05.

Multiple regression of oyster weight, length, age, and infection with
P. marinus on the magnitude of infection (log CFU [g.sup.-1]) of
V. vulnificus.

Variable DF Regression Error P

All variables 4 -1.258 0.396 0.021 *
Weight 1 0.007 0.005 0.002 **
Length 1 -0.032 0.148 0.129
Age 1 0.005 0.148 0.765
P. marinus 1 0.975

* = P < 0.05; ** = P < 0.01.
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Author:Oliver, J.D.
Publication:Journal of Shellfish Research
Geographic Code:1U5NC
Date:Aug 1, 2005
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