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Evaluation of three northern quahog (= hard clam) Mercenaria mercenaria (Linnaeus) strains grown in Massachusetts and New Jersey for QPX-resistance.

ABSTRACT Three selected aquaculture strains of the commonly used hard clam, Mercenaria mercenaria (Linnaeus, 1758)--Massachusetts (MA), New Jersey (N J), and South Carolina (SC)--were reared in a single hatchery. They were planted in replicate plots in Barnstable Harbor, MA, and Dry Bay, N J, in spring 2008 and grown to market size. Growth, survival, and QPX (quahog parasite unknown) prevalence and severity were measured in fall 2008, spring 2009, and fall 2009. Growth was similar at both sites for the first summer, but during the second year growth was better in Massachusetts. Overall survival was better for all strains in New Jersey when compared with Massachusetts. Survival of strains in New Jersey was 53.4% and 34.8% for NJ and MA, respectively, closely followed by those in Massachusetts (41.8 % and 26.2% for NJ and MA, respectively). Strains from SC seed had the highest prevalence and severity of QPX and the lowest survival (36% and 6.6% in New Jersey and Massachusetts, respectively). Infections with QPX were low at both sites, but this study confirms earlier work indicating that QPX infection rates appear to be strain specific, with strains of southern origin being more susceptible than those of northern origin. Our study also supported anecdotal reports that QPX disease is more severe in Massachusetts than in more southern sites where it has been found. As in most previous field studies, we found that although mortality was correlated with QPX levels, it was considerably higher than infection prevalence would indicate, which suggests that strain interactions with stressful environmental conditions or unidentified factors may be also involved in mortality.

KEY WORDS: QPX, hard clam, northern quahog, Mercenaria, disease resistance

INTRODUCTION

The hard clam Mercenaria mercenaria is relatively unique among cultured bivalves in that all aquacultured seed are hatchery produced, because wild seed are not sufficiently abundant to satisfy aquacultural demand. This makes the production of seed and the strains being used a critical management decision for hard-clam aquaculturists. QPX (quahog parasite unknown) has been responsible for significant losses of cultured and wild strains of hard clams from Massachusetts to Virginia. In cultured clams, the parasite is acquired after planting, and there is no evidence that hatcheries are the source of the pathogen (Ford et al. 1997). Although most outbreaks of QPX disease have been reported in aquacultured clams, the disease also occurs in populations of wild clams (MacCallum & McGladdery 2000, Dove et al. 2004).

The greatest number of severe disease outbreaks has been reported in Massachusetts, and neither the parasite nor the disease has been reported south of Virginia. Massachusetts experienced a QPX outbreak in Provincetown in 1995 that essentially ended hard-clam culture at that site (Smolowitz et al. 1998). Aquacultured strains in Barnstable Harbor suffered severe mortality from QPX after its presence was first confirmed in 2001, and its presence there has continued (Lyons et al. 2007). An outbreak in cultured hard clams in Wellfleet resulted in the removal and destruction of clams from all plots in 2004.

In New Jersey, growers experienced significant (up to 80%) losses between 1995 and 1998, when southern (South Carolina, and possibly Florida strains) seed was used, but similar losses were not found in local (New Jersey (N J) strain) seed planted nearby (Ford et al. 2002). These losses were essentially eliminated by not using these southern strains, thus saving the industry from the associated economic loss. An outbreak of QPX in a dense set of wild clams in the Raritan Bay estuary of New Jersey and New York occurred in 2002, and regulatory agencies in both states stopped relay programs to prevent transfer of the parasite (Dove et al. 2004).

These losses have fostered an ongoing series of investigations into the disease and its etiology. Evidence that QPX susceptibility in cultured animals is related to the strain's genetic background was first provided by Ford et al. (2002) and was subsequently confirmed by additional field studies (Ragone Calvo et al. 2007, Dahl et al. 2010). The study by Ragone Calvo et al. (2007) compared the growth and mortality of 5 strains of hard clams (Florida (FL), South Carolina (SC), Virginia (VA), and NJ, and Massachusetts (MA)) produced in the same hatchery, and planted at commercial densities in replicate plots in Virginia and New Jersey. In their study, susceptibility to QPX was related to the strain's geographic (and presumed genetic) origin. By the end of the experiment (approximately 3 y) the southern strains generally grew faster at both sites than more northern strains, but mortality was higher. Based on the numbers harvested at the conclusion of the study, total mortality was 78%, 52%, 36%, 33%, and 20% for FL, SC, VA, NJ, and MA strains, respectively, in Virginia; and 53%, 40%, 20%, 6%, and 4% for the same strains, respectively, in New Jersey. Dahl et al. (2010) provide evidence that infection of a FL strain occurred within 2 mo after deployment in New York. A NY "wild type" and a NY "notata" strain were also deployed. The wild-type seed did not become detectably infected, and the notata seed were infected during the second summer. Both of the NY strains had higher survivorship than the FL strain. These data generally support the strain x latitude interaction found in the prior studies, but they do not permit documentation of the anecdotal reports that for a given strain, mortality is greater in Massachusetts than in New Jersey or Virginia. In the study by Ragone Cairo et al. (2007), the clams deployed in Massachusetts were lost to ice during the first winter, and the data from New Jersey and Virginia did not support a south-to-north latitudinal gradient of increasing infection severity, suggesting an independent environmental variable. Documentation of the effect of latitude and strain was still lacking in Massachusetts, the area that appears to have shown consistently the highest levels of infection and mortality. The current study was designed to investigate the possible latitudinal component of QPX disease utilizing the same strains that were used in the study by Ragone Cairo et al. (2007).

MATERIALS AND METHODS

Seed from populations of the same strains that were used in the study by Ragone Cairo et al. (2007) were reared at the Virginia Institute of Marine Science and grown to planting size (15-18 mm) on the eastern shore of Virginia. We deployed 3 strains (MA, NJ, and SC in origin) in spring 2008 in Dry Bay, N J, and Barnstable Harbor, MA both sites with ongoing hard-clam culture. The planned fall 2007 planting was delayed because histological analysis of the seed (100 from each strain) prior to shipment found a previously unknown and rare haplosporidian parasite, which was subsequently described (Ford et al. 2009). Two subsequent samplings of equal size in spring 2008 showed no parasite presence, including QPX. There were no unusual overwintering mortalities, and animals were cleared for shipping to the test sites.

This additional winter of holding the seed resulted in fewer clams than had been anticipated in all groups as a result of overwinter mortality. Clams were allocated by first counting and weighing a subsample, then dividing the total numbers of clams in a strain into 8 equal-weight portions. Because of the overwinter mortality, unequal numbers of clams from each strain were available for planting (approximately 990,723, and 1,005 per plot for the N J, MA, and SC strains, respectively): but for each strain, equal numbers were planted in Massachusetts and New Jersey. Four replicate plots of 1.52 x 1.52 m on each side (2.31 m2) were established for each strain at each site in a general randomized block design. Lots of clams were assigned to each plot in a random fashion. Experimental densities approximated commercially planted densities.

Plots, planted in spring 2008 were sampled in spring 2009 (April/May) and either the late summer or fall (September to November) during 2008 and 2009 depending on when QPX was expected to become prevalent at each site. At both sites, sampling within each plot was by random selection of core locations based on a grid that was placed over each plot. Ten samples were collected from each plot using a 10.2-cm-diameter corer that was forced into the sediments to a depth of about 15 cm. In New Jersey, the clams and substrate were removed and placed in numbered plastic bags. The bags were transported to the laboratory, retained in a cold room overnight, and then clams were removed from the sediment by sieving on a 2-mm mesh sieve. In the second year, a 4-mm mesh was used. At the Massachusetts site, all cores were sieved in the field through a 4-mm mesh and the clams transported to the laboratory for processing. All live and dead clams, including single shells, were retained for counting and measuring, and individuals were set aside for histological processing and subsequent microscopic examination. On the final sampling date, cores were taken to allow for comparisons with earlier sampling, and then the entire plot was dug either by bull rake and hand (New Jersey) or bull rake (Massachusetts). This latter sampling provided a means to estimate the efficiency of the overall sampling effort and to estimate overall mortality.

Data analysis used standard statistical tests (ANOVA for growth and/or chi-squared contingency tests of independence for mortality) within each sampling date. The final sampling provided an overall estimate of growth and survival. All data analysis of survival was based on adjustments that assumed an equal number of each clam strain planted in each plot.

Gross and histological evaluations of 15 clams from each replicate plot were made for each sampling date (60 clams per strain per time, 1,080 total for both locations). All animals were measured (length, width, and height) and shucked. A cross-section taken from the hinge region through the visceral mass to the mantle edge, and a piece of the mantle adjacent to the siphon, were fixed in Davidson's AFA for 48 h. At initial deployment and in fall 2009, a piece of the clam, including viscera and foot, was collected, placed in 70% EtOH, and archived at 80[degrees]C for future genetic analysis. After collection, clams were processed to fixation within 96 h. Fixed tissues were processed into tissue slides and examined at the Haskin Shellfish Research Laboratory of Rutgers University according to standard histological procedures (Smolowitz et al. 1998, Ford et al. 2002). Infections, when present, were evaluated for both parasite abundance and distribution using a system modified from Ford et al. (2002). The number of viable and apparently nonviable parasites in the entire section was estimated as rare (1 10), light (11 100), moderate (101-1,000), or heavy (>l,000), and was scored 1-4 in that order. The distribution was categorized as focal (single lesion or infection site), multifocal (multiple lesions or infection sites), or diftuse (parasite distributed throughout host tissues) and scored 1 3, respectively. The location and number scores were multiplied to provide an index to the severity of infection in each individual. An average of all severity values, including 0s, was calculated for each strain at each site for each sampling date.

The prevalence data were arcsine transformed and analyzed by ANOVA. The mean severity data were analyzed by chi squared (Snedacor & Cochran 1956). Two clams (one each from the SC and NJ strains) from Massachusetts in fall 2008 were found with only moribund QPX in the tissue. These were considered to be uninfected when the data were analyzed.

We also recorded the numbers of the non-QPX organisms found in histological sections. These were placed in 5 categories: Rickettsiales/Chlamydiales-like organisms (R/Ch), Trematoda, Ciliata, Cestoda, and neoplasia.

RESULTS

Growth

For the first 3 sampling periods, which spanned winter 2007/ 2008, the clams were held in Virginia (Fig. 1). At the time the clams were shipped and planted in March 2008, mean shell lengths ranged from 14-18 mm, with SC > MA > NJ (Fig. 1). Clams of all strains at least doubled in length to more than 30 mm by the first postdeployment sampling in fall 2008. At that time, SC clam strains in New Jersey were larger than all clams in Massachusetts and the NJ clam strain in New Jersey (Fig. 1). All clams in Massachusetts were the same size. By the spring sampling in 2009, all strains in Massachusetts were still the same size, but were larger than those in New Jersey, and these in turn were all the same size. By the final sampling in the fall 2009, all clam strains in Massachusetts were larger than those in New Jersey. In Massachusetts, the MA strains were larger than SC strains, and the NJ strains were between the other two strains and did not differ from either of them. At the final sampling, all strains in NJ were statistically the same and had reached market size of about 45 mm in shell length (Fig. 1).

[FIGURE 1 OMITTED]

Survival

We compared survival in two ways, first by collection date based on live/dead counts in core samples, and second by counts of live clams harvested from the plots at the end of the experiment (Fig. 2). Repeated-measures ANOVA of arcsine-transformed live/dead animals from core samples randomly allocated within each plot found significant differences in numbers of individuals per core between strains, collection dates, and an interaction between site and collection date (P = 0.0037, 0.0099, and 0.0199, respectively). Because of the interaction, variability between plots, strains, and dates in the repeated-measures analysis did not show differences between sites. Analysis of harvest data (discussed later) clearly indicated site and some strain differences. The mean number per core for clams from all plots pooled over both sites was 3.9, 3.3, and 2.6 for N J, MA, and SC strains, respectively. The NJ and SC strains were significantly different from each other, and the MA strain was between, but not significantly different from them. Collection date differences were the result of the first sampling date having more clams per core (4.0) than the following two dates (2.9 and 2.9). The interaction between site and collection date was primarily the result of a large number of clams (4.3) per core being collected at the Massachusetts site during the first collection period relative to the following two collections (2.9 and 2.1, respectively), which were lower and not different from each other.

Because of the interaction and because of obvious differences in survival when the plots were harvested (Table 1), we analyzed the core data from each site separately. At the Massachusetts site there were significant differences resulting from collection date (P = 0.0000) and date x strain interaction (P = 0.0333). At the first sampling, more clams were found than in the following two sampling periods, which were not different. The final core data showed differences between sites (P = 0.0152). There were no differences between strains in New Jersey (P = 0.6937), but in Massachusetts the SC strain was significantly different from the NJ and MA strains, which were not different from each other (P = 0.0068).

[FIGURE 2 OMITTED]

After the final core sampling, the plots were completely harvested and these data were used to estimate survivorship (Table 1). ANOVA of the harvest data showed differences in numbers between the sites (P = 0.0000) and strains (P = 0.0003). Overall numbers of clams surviving in each strain, and thus percent surviving, were greater in New Jersey than in Massachusetts (Fig. 2). Numbers of MA strains in New Jersey were not significantly different from the NJ strain in Massachusetts, and the numbers of SC strains in New Jersey were not significantly different from the numbers of NJ and MA strains in Massachusetts (Table 1, Fig. 2A). Based on the final sampling, the significant differences in the harvest data are the same as with the core data.

Comparison of numbers per core at the final collection date normalized to core area and extrapolated to the total plot area showed significantly fewer animals relative to those collected when the total plot was harvested (chi squared = 40.49, df = 22). This was primarily the result of differences in Massachusetts. Whether these are the result of differences in collection of small, dead individuals in the core samples or the result of incomplete harvest cannot be determined from the data. If harvest was incomplete, and results in Massachusetts are based on core data only, survival of the MA and NJ strains would have been as good as or better than in New Jersey (Fig. 2B).

Histology

Samples taken prior to planting showed no QPX in any of the strains, and QPX prevalence found in subsequent samples was consistently less than 20% (Fig. 3). Nevertheless, prevalence of QPX was significantly higher (ANOVA, P = 0.0022) in Massachusetts than in New Jersey, and at both locations was generally higher in the SC strain (P = 0.0001) than in the MA or NJ strains, which were not significantly different from each other. Prevalence of only 2 samples (SC strains in Massachusetts from the two fall samplings) was significantly different from all others (Fig. 3). Infection severity was also relatively low during the study period (Fig. 4). Chi-squared analysis of ranked data (Wilcoxon 1949) indicated no heterogeneity (chi squared = 34, n = 9). Thus, the data at each site vary in a similar manner. Further analysis of unranked data indicated no differences between the sites (chi squared = 0.97, df = 9). Separating the data by site also yielded no significant within-site heterogeneity (Massachusetts: chi squared = 0.49, df = 4: New Jersey: chi squared = 0.75, df = 4). The infection severity data were then compared against a hypothesis that the distribution of the readings should be equal for all times, sites, and strains. The chi-squared values again indicate no significant differences (chi squared = 5.09 and 0.90 in Massachusetts and New Jersey, respectively). The highest deviation expected was for the 2 fall samples of the SC strain in Massachusetts, but neither value was significantly different from the others.

[FIGURE 3 OMITTED]

Of the other parasites (R/Ch, Trematoda, Ciliata, Cestoda and neoplasia) found in histological sections, we only found 1 incident of the latter two categories. There were generally higher levels of the abundant parasite classes in New Jersey relative to Massachusetts. Combining these data and testing for heterogeneity with chi squared at the P = 0.05 level indicated significant heterogeneity. We then tested each of the areas separately and, with the exception of the predeployment data from Wachapreague (no significant heterogeneity, but significantly elevated R/Clo in the SC stock), there was significant heterogeneity for combined parasite load in both New Jersey and Massachusetts. Within both of these sites there was also significant heterogeneity if the combined data are analyzed by collection period. The final analysis was to compare each of the parasite loads for an individual parasite groups by collection period. For the Massachusetts site there were significantly more R/Ch organisms in the MA strains in the fall 2008 sampling, and no differences for any of the parasite groups for the spring 2009 and the fall 2009 collections. In New Jersey, the same analysis found more R/Ch in the MA strain from the fall 2008 sampling, fewer ciliates in the SC strain in the spring 2009 sampling, and significantly more ciliates in the MA strain in fall 2009. Most of these were found in low abundance in the histological sections. At these levels they are not known to cause mortality, and we do not believe there was any significant interaction with QPX. The generally greater parasite loads at the New Jersey site, where mortality was less, would generally support the lack of significant interaction.

[FIGURE 4 OMITTED]

DISCUSSION

QPX is a naturally occurring organism, and in environmental samples it has been found associated with a wide variety of substrates. Lyons et al. (2005) reported the presence of QPX on marine aggregates, and Gast et al. (2008) examined water, sediments, macroalgae, invertebrates, and sea grasses, and found QPX in all sample types in Massachusetts, but only in algae, sediments, and invertebrates in Virginia. In addition, in Massachusetts they found a seasonal pattern to the occurrence of QPX that differed between sample type, with higher values for seawater and algae in spring, and higher values for invertebrates in the fall. The QPX organism appears to be restricted to euhaline and mesohaline salinity. Data from cultured QPX evaluation of salinity tolerance showed very low growth at salinities at or below 20 psu (Brothers et al. 2000). Perrigault et al. (2010) reported survival of QPX maintained in artificial seawater at 15 psu, but no growth in culture medium of the same salinity.

QPX has caused significant losses to hard-clam aquaculturists in Virginia, New Jersey, and Massachusetts. There is no evidence that clams become infected in the hatchery; rather, they are typically infected after they are planted in grow-out locations (Ford et al. 1997). In addition to the current study, three field studies provide significant evidence that QPX susceptibility is linked to particular strains of hard clams (Ford et al. 2002, Ragone Calvo et al. 2007, Dahl et al. 2010), and it is likely that these are linked to genetic differences, but reports of disease outbreaks in wild populations (Dove et al. 2004) suggest an environmental component as well. The current study and the studies by Ford et al. (2002), Ragone Calvo et al. (2007), and Dahl et al. (2010) indicate that planted seed from strains that originate at geographically different latitudes have significant differences in their susceptibility, and strains originating in the south perform poorly at more northern sites. There is mixed evidence for a strain-independent, latitudinal shift in QPX-related mortality. The study by Ragone Calvo et al. (2007) found greater infection levels and higher mortality in Virginia plantings than in New Jersey when individual strains were compared between locations (Table 2). Anecdotal evidence and the current study have shown that QPX infections were greater in Massachusetts than in New Jersey (Table 2). It is important to note that both the Virginia portions of the study by Ragone Calvo et al. (2007) and the Massachusetts portion of the current study were both in the intertidal zone, whereas the New Jersey site for both studies was rarely, if ever, completely exposed. The exposure may provide either the added stress necessary for infection, or may cause the clams to remain closed for extended periods, thus allowing more time for QPX in materials retained in the mantle cavity to infect the clam. The study by Ford et al. (2002) included an intertidal plot in New Jersey, but was substantively different from the other studies in that most of clams that were assigned to plots were already infected. In addition, these clams were not planted as seed, but were animals that had been grown on the site for about 1.5 y.

There is also evidence that QPX is primarily a coldwater disease because it has never been found south of Virginia. The results from studies of cultured QPX showed that optimum in vitro growth was at 20 23[degrees]C and that mortality of all QPX occurred at a temperature of-32[degrees]C (Brothers et al. 2000, Bugge & Allam 2005, Perrigault et al. 2010). Dahl et al. (2008) used laboratory cultures of QPX to infect seed clams from Massachusetts, New York, Virginia, and Florida, and found that southern seed (FL and VA) were more susceptible to infection, thus mirroring the field results of Ragone Calvo et al. (2007). In addition, different QPX isolates had differing levels of virulence (Dahl et al. 2008). Although hard clams are susceptible to QPX, they do have some mechanisms of defense, and these mechanisms apparently are tissue specific. Extracts from different clam tissues had different inhibitory effects on the growth of cultured QPX (Perrigault et al. 2009), but these defenses may be reduced in the presence of stress-inducing factors such as harmful algae (Hegaret et al. 2010). Thus, field studies such as that of Ragone Calvo et al. (2007) and our study are in accord with the laboratory work in that QPX infection pressure and disease prevalence are generally greater as one moves from Virginia to the north, and that strains of clams that are derived from southern stocks are more easily infected when grown in the north. Whether this increased infection rate is the result of genetically derived resistance to QPX or the result of increased stress on southern strains when they are grown in northern locations, or an interaction between the two is unknown, but Dahl et al. (2010) found FL strains planted in the summer were infected within 2 mo. They suggested that this, at least in part, negates the suggestion that cold temperatures are the environmental stress that causes southern strains to be more prone to infections in the north. The data from the current study augment those of the prior field studies (Ragone Calvo et al. 2007, Dahl et al. 2010), indicating that QPX infects different strains at different rates and that strains derived from stocks occurring from Virginia south are more susceptible to infections than those derived from New Jersey or Massachusetts populations. Although infection levels in all strains were relatively low in the current study compared with those of the studies by Ragone Calvo et al. (2007) and Ford et al. (2002), infection prevalence was still highest overall in the SC strains.

Although the link between QPX presence and poor survivorship has been made in a number of studies, including ours, there is still no definitive study indicating that QPX is responsible for all the associated mortality. The differential prevalence in different strains planted at the same site may indicate a stress-related mechanism that allows QPX to establish infections. It is also important to note that the mortality is typically much higher than prevalence level would suggest. As with the study by Ragone Calvo et al. (2007), infection rates in New Jersey during the current study were low (never reaching 10%). and infection severity was also low, yet total mortality was between 46% and 64%. This disparity between infection and mortality levels leads to at least three potential explanatory hypotheses: First, QPX develops very rapidly from infection to mortality, and our sampling schedule was not frequent enough to detect the link. Second, the mechanism of QPX-induced mortality can be expressed at infection intensities lower than those we were able to detect histologically. Third, QPX is a secondary invader that may cause some mortality when it proliferates to high levels, but the underlying stressor/pathogen has yet to be identified.

Given the number of samples in the current study and that of Ragone Calvo et al. (2007) that have shown a pattern of relatively high mortality associated with low QPX prevalence and intensity, it seems unlikely that the first hypothesis could be true, but no field studies have definitively described the establishment and progression of infections in newly exposed clams using closely timed sampling. Dahl et al. (2010) did find relatively high QPX prevalence in the FL strain, but mortality was as high or nearly as high in NY strains that had low or no QPX prevalence. Most QPX studies have used bimonthly or spring/fall sampling schemes, and there is a need to sample at least monthly, if not on the order of every 2 wk, to describe definitively the onset and development of infections, subsequent seasonal patterns of potentially chronic infections, and mortality. For instance, monthly monitoring of Eastern oysters, Crassostrea virginica, has permitted definitive descriptions of seasonal cycles of the pathogens Haplosporidium nelsoni (MSX) and Perkinsus marinus (Dermo), demonstrated latitudinal shifts in the cycles, and clearly linked infection to mortality (Ford & Haskin 1982, Bushek et al. 1994, Ford & Smolowitz 2007). Given the results of in vitro studies showing a temperature dependence on both growth and survival, it is likely that a seasonal infection pattern exists for QPX as well, but it is difficult to identify one clearly from published reports, which show infection peaks variously in the spring, summer, or fall. Dahl et al. (2010) show clearly a spring/summer infection period. Ragone Cairo et al. (2007) could not identify clearly a seasonal infection cycle, but the data also suggest a spring/ summer onset of infection. Our study suggests a spring/summer infection period, but sampling was too infrequent to assign this definitively to spring or summer. Whether the disparities in infection onset and peaks are linked to the strains being tested or the location of the test is unclear, but the inhibitory effect of high temperatures found in vitro (Brothers et al. 2000, Perrigault et al. 2009, Perrigault et al. 2010) suggests that it is possible that in more southern areas of the known range of QPX, such as New Jersey and Virginia, periods of high temperature may retard QPX development, but there is no field evidence to support such speculation.

Only one study (Ford et al. 2002) has described QPX infection prevalence and severity in dead and dying clams. Prevalence was 86-100% compared with about 50%, and severity was 2 3 times greater, in dead and dying clams than in live clams collected at the same time. The study by Ford et al. (2002) used highly susceptible SC clams that became more heavily infected with QPX than did those in the current study, but it would be highly unusual if more resistant clams such as the NJ and MA strains used in the current study were killed by infections so light or focal that they were not detected histologically. Instead, the data available indicate that lethal QPX infections can be detected histologically, which is counter to the second hypothesis.

The third hypothesis is at least consistent with what is known about thraustochytrids being opportunistic invaders, but whether it is the QPX, another stressor, or some combination of the two that causes the mortality deserves investigation. Consistent with the findings of Ford (2001 ) of the low incidence of pathogen-induced mortality in hard clams relative to the Eastern oyster C. virginica, no other pathogens have been reported in the QPX field studies. Mortality levels of aquacultured hard clams often reach or exceed 30% within the first year after planting, and most of this is presumed to be caused by predation or "'winter mortality," but no definitive studies have ever been conducted to separate the two. The field studies of QPX have all reported mortality prior to infection by QPX, but only two field studies report on the timing of the mortality. Ragone Calvo et al. (2007) reported 15-40% mortality in both the Virginia and New Jersey experimental plots prior to observed QPX infection. In Virginia, the mortality observed during the first sampling period did not show an increase in any strains for nearly a year (the second spring season sample), even though some were infected with QPX in the prior spring. There was a slight, nonsignificant increase in mortality during the second spring, followed by a decrease during summer and an increase in the fall. In New Jersey, southern strains (FL, SC, VA) all experienced increased mortality after the replanting in the spring and fall, but mortality in the northern strains (MA and N J) remained the same level after the initial mortality (Ragone Calvo et al. (2007). At both sites, mortality and QPX were positively correlated after QPX prevalence and intensity began to increase.

In our study, the initial mortality at the New Jersey site mirrored that of the work by Ragone Calvo et al. (2007) and was nearly 40% for all strains. At this site there was little QPX infection, and mortality increased only slightly for the remainder of the study. At the Massachusetts site, initial mortality did not exceed 25% between the spring planting and fall sampling, but increased to 25% to more than 40% by the following spring. Mortality during the summer and early fall remained at the spring levels except for the lightly QPX-infected SC strain, in addition, the marked decline of both prevalence and infection severity in this strain from fall 2007 to spring 2008 (Figs. 3 and 4) coincided with an increase from 22-53% in mortality (the greatest increase measured during the study) over the same period (Fig. 2B). Whether this is the result of the infection or an infection plus winter stress cannot be ascertained from the current study.

The initial mortality may be cases of "overwinter" mortality or predation, both of which primarily affect smaller seed. This is presumed to be the result of starvation (Zarnoch & Schreibman 2008) and/or bacterial infection (Kraeuter & Castagna 1984), and may be more severe in some strains (Bricelj et al. 2007). In general, this overwinter mortality lessens as the clams become larger. In cultured clams without documented QPX, total mortality for the 2 3 y required for planted seed to reach market size ranges from 30-90%, with "typical" losses in the 40 60% range. These ranges are consistent with what was experienced for the MA and NJ strains in New Jersey during the current study. Mortality in Massachusetts was higher than these ranges for all strains, but especially the SC strain. Although mortality levels for the culture period can be approximated, it is assumed that QPX-associated mortality would be in addition to this "background" level, but the lack of data on what causes the "'background" makes definitive statements imprudent.

Even in the absence of highly susceptible southern seed, there have been many more reports, both documented and anecdotal, of heavy QPX-associated mortality in Massachusetts than in other areas of the northeast (Smolowitz et al. 1998, Lyons et al. 2007). This suggests that conditions in Massachusetts may lead to higher infection prevalence and severity levels than at other sites. For instance, the presence of externally visible nodules along the mantle edge, which contain masses of QPX, are relatively common in Massachusetts, but have rarely been reported in New Jersey or Virginia (Ragone Calvo et al. 1998, Ford et al. 2002, Ragone Calvo et al. 2007). Our study is the first that has successfully deployed and followed the same strains at the same time in both Massachusetts and New Jersey. In Massachusetts, both the prevalence and severity of infections were higher than in New Jersey, lending support to the contention that QPX-induced mortality increases with latitude. One potential environmental inducer of QPX infection in Massachusetts that was not encountered in New Jersey is that the Massachusetts clams were planted in the intertidal zone, whereas in New Jersey they were planted in an area exposed only on the most extreme of low tides that coincide with strong westerly winds. The intertidal location may be the additional factor that, when coupled with the high density, facilitates QPX infection. The intertidal location would normally prevent the clams from feeding, and thus any pseudofeces that accumulated in the mantle cavity might remain in contact with the tissues for longer periods. The increased contact coupled with lack of ventilation may be a factor in allowing QPX to invade the tissues. This is also consistent with the study by Ragone Calvo et al. (2007) in which the clams exposed at the Virginia site attained higher QPX prevalence and intensity, and higher mortality than did the clams exposed in New Jersey. The Virginia site was higher in the intertidal than the New Jersey site. The latter, although different from the New Jersey site used in the current study, was only exposed on extreme spring tides.

The better growth in Massachusetts relative to New Jersey starting in the spring of 2009 and continuing throughout that growing season could be the result of a number of factors. Better survivorship in New Jersey certainly caused the clams to be denser, but the N J-strain clams in Massachusetts were nearly the same density as the SC clams in New Jersey, and growth was still better in Massachusetts. This comparison is obviously compromised because although the density was the same, we are comparing growth of different strains. It is also possible that the clams experienced different food levels or quality at the two sites. The higher mortality rate in Massachusetts may have left larger survivors, thus making it appear as if growth was greater, but growth data are based in animals collected in the cores, and both growth and size were significantly greater in Massachusetts from fall 2008 to spring 2009, and mortality was no higher than in New Jersey (Figs. 1 and 2B). The rate may reflect the earlier fall sampling time in Massachusetts, but the size attained by spring the following year was greater in Massachusetts for all strains. Another factor is that Dry Bay, the New Jersey site, is densely planted with commercial clams so it may be that the culture area is reaching its carrying capacity. The Massachusetts site had few areas of high-level commercial activity nearby and thus there should have been limited influence on food supplies reaching the experimental plots. There is evidence from Dry Bay that at times of low water in the summer, dissolved oxygen levels can drop to less than 2.5 rag/L, and this, too, would reduce growth--at least temporarily. Although we do not have information directly from the Massachusetts site, summer daytime dissolved oxygen from the nearby Barnstable Harbor dock never went below 6 rag/L, so it is unlikely that it would cause a growth reduction. Although our studies did not evidence very high levels of QPX infection, we set aside samples for future genetic analysis so that this work could proceed without conducting another field study. The genetic link between QPX susceptibility and particular strains has not been subject to genetic analysis. Using AFLP markers and maps, Guo and colleagues (Yu & Guo 2003, Guo et al. 2004, Yu & Guo 2006) have shown that disease resistance in the Eastern oyster can be mapped quickly by identifying and mapping markers that show significant frequency shifts after disease-caused mortalities. Given the time and expense of developing and sampling field trials, it seems prudent that when such studies can provide useful information for breeding programs, taking samples for genetic analysis at the beginning and end should be part of most protocols.

ACKNOWLEDGMENTS

This study could not have been completed without the help of several other individuals. In particular, we thank Iris Butt and Emma Green-Beach of Haskin Laboratory for assistance with sampling and/or sample processing. In Massachusetts, we had help in the field from the BARS (Barnstable Association for Recreational Shellfishing) group and CCCE marine program specialist, Joshua Reitsma. The manuscript was greatly improved by the comments and insights of an unknown reviewer. The study was funded by a grant from the Northeast Regional Aquaculture Center.

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JOHN N. KRAEUTER, (1) * SUSAN FORD, (1) DAVE BUSHEK, (1) EMILY SCARPA, (1) WILLIAM C. WALTON, (2) DIANE C. MURPHY, (3) GEF FLIMLIN (4) AND GEORGE MATHIS (5)

(1) Haskin Shellfish Research Laboratory, Rutgers University, 6959 Miller A venue, Port Norris, N J; (2) Auburn University Shellfish Laboratory, 150 Agassiz Street, Dauphin Island, AL; (3) Cape Cod Cooperative Extension & Woods Hole Sea Grant, P.O. Box 367, Barnstable, MA; (4) Ocean County Ag. Center, 1623 Whitesville Road, Toms River, N J; (5) Mathis and Mathis Inc., 143 Leektown Road, Egg Harbor, NJ

* Corresponding author. E-mail: kraeuter@hsrl.rutgers.edu

DOI: 10.2983/035.030.0321
TABLE 1.
Mean numbers of hard clams collected at harvest from
experimental plots in Massachusetts and New Jersey
for 3 strains (MA, NJ, and SC).

                         Mean No.           95% Confidence
Site            Strain   per Plot    SD         Limit

New Jersey        MA     418 (ab)   82.22       176.91
                  NJ     534 (a)    24.46        52.63
                  SC     364 (bc)   16.47        36.02
Massachusetts     MA     261 (c)    53.61       115.35
                  NJ     349 (bc)   80.74       173.71
                  SC      66 (d)    20.63        44.38

All data were normalized to a base of 1,000 clams planted in each plot
(n = 4 plots per strain) and animals removed during sampling. Numbers
with the same lowercase letters are not significantly different.
Percent survivorship can be obtained by dividing the number of
individuals per plot by 10.

TABLE 2.
Mortality and QPX infection prevalence in hatchery grown
strains of Mercenaria mercenaria planted and grown to
market size at field sites.

                       Ragone Calvo
                       et al. (2007)

              New Jersey           Virginia

Strain   Mortality    QPX     Mortality   QPX

FL          53         18        78       29
SC          40         38        52       21
VA          20         18        36       10
NJ           6          5        33        2
MA           4          0        20        0

                      Current Study

              New Jersey       Massachusetts

Strain   Mortality    QPX     Mortality   QPX

FL
SC          64         7         93       18
VA
NJ          47         2         65        3
MA          58         0         74        2

"Mortality," final percent mortality based on harvesting of entire
plots; "QPX," maximum percent prevalence observed during the study.
See text for strain identification.
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Author:Kraeuter, John N.; Ford, Susan; Bushek, Dave; Scarpa, Emily; Walton, william C.; Murphy, Diane C.; F
Publication:Journal of Shellfish Research
Article Type:Report
Geographic Code:1USA
Date:Dec 1, 2011
Words:7697
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