A REVIEW OF QPX DISEASE IN THE NORTHERN QUAHOG (= HARD CLAM) MERC EN ARIA MERCENARIA.
Drinnan and Henderson (1963) first reported a disease causing high mortality in wild hard clams in Neguac and Malpec in the Gulf of St. Lawrence, NB, Canada. They noted up to 80% mortality in dense populations of wild clams. At that time, the organism causing the disease was classified morphologically as a chytrid fungus. In 1994, Whyte et al., reported a hard clam disease caused by a protistan parasite, which they named Quahog (Quahaug) Parasite Unknown (QPX), causing major mortality in a hatchery/nursery setting in small (15-30 mm shell height) clams on Prince Edward Island, Canada. Histological examination of the infected clam tissue identified the same organism as reported in the previous epizootic.
Hard clam aquaculture in the United States has historically been free of significant diseases causing morbidity and mortality; however, in 1995, high morbidity and mortality were noted in 1.5 to 2-y-old cultured hard clams in Provincetown and Duxbury, MA. Aquaculturists had experienced annually increasing mortality in their submarket-sized hard clams over the 3 y since initiating hard clam culture in the Provincetown Harbor. In 1995, mortality was estimated by culturists and researchers at 80% on some aquaculture sites (Smolowitz et al. 1998). Examination of the affected clams, submitted to R. Smolowitz by aquaculturists from Provincetown in early July 1995 showed small gaps between valves resulting from the lack of closure of free valve edges. Also prominent were small, V-shaped chips in the shell edges and increased mucus and entrapped sand particles along the exposed mantle edges along the affected valves. Quahog Parasite Unknown was identified in 71% of the clams examined histologically (Smolowitz et al. 1998).
During a visit to the intertidal aquaculture sites in Provincetown, MA, at low tide later that month by D. Leavitt and R. Smolowitz, a crunching and grinding noise was noted in infected, recently harvested clams that had been placed into collection baskets. Most interestingly, in affected leases in sandy sediment sites, live and dead clams were lying in large numbers on the sediment surface (Fig. 1). Replicate cores collected by Leavitt and Smolowitz in October 1995 were used to determine the extent of mortality in two affected locations in Provincetown and showed mortality of 35.7% in one site and 20.6% in another site (Smolowitz et al. 1998). Other sites in the harbor were also affected but not as dramatically. Irregular yellow, firm swellings and nodules of 1-4 mm were noted in the mantle edges and membranous mantle, especially adjacent to the siphon, in many of the affected clams. Rarely, the nacre of the shell underlying the nodules was eroded. Histological examination of live clams from the core samples taken from the sites showed a prevalence of 90% of animals. A second Harbor in Duxbury, MA, similarly reported clam mortality from QPX a few months later (Smolowitz et al. 1998). Identification of this disease of hard clams in the United States (Smolowitz, et al. 1998) alarmed many aquaculturists and researchers along the U.S. eastern coast.
QUAHOG PARASITE UNKNOWN IN THE UNITED STATES BEFORE 1995
Interestingly, retrospectively, an organism identified in severely necrotic tissues of a few hard clams submitted for histological evaluation from a mortality event in the Mitchell River, Chatham, MA, in 1992 was determined to be QPX. At the time of that die off, the shellfish warden noted the subtidal area was densely populated with wild hard clams. Follow-up investigations a few months later in 1992 revealed that only a few clams were still present in the area and histological examination of animals sampled at that time detected no infected clams. Another mortality incidence occurred in wild hard clams in an area of Barnegat Bay, NJ, in the 1960s. The infectious organism was not named at the time of the event. On examination of the archived materials, Ford et al. (2002) reported that the epizootic was due to QPX. Such retrospective findings illustrate that sometimes "new" diseases have been present for extended time periods but not yet well identified.
QUAHOG PARASITE UNKNOWN OCCURRENCE AFTER 1995
From 1995 to 2006, QPX disease has occurred in epizootic proportions in other sites in Massachusetts, including Barnstable Harbor and Cotuit Bay in Barnstable, and Pleasant Bay in Orleans and Wellfleet Harbor in Wellfleet. Samples of animals lying on the surface of the sediment in several of the aquaculture raceways in Barnstable showed severe infection. For example, in one location in December 2001 prevalence in surface animals was 100%, and 83% of those animals contained macroscopically visible mantle nodules; samples of animals buried in the underlying sediment showed a 12% prevalence with only 2% containing macroscopically visible mantle nodules. Other aquaculture sites in the same area were not affected at that time. Interestingly, in the following years, the disease spread throughout the aquaculture sites in Barnstable Harbor (Smolowitz, unpublished survey data). These findings strongly suggested that the disease was contagious.
In 2002, QPX was identified in 7% of large adult wild clams that were to be relayed from Palmer Island, New Bedford, MA, to locations on Cape Cod, MA (Smolowitz, unpublished survey data). In 2004, QPX was first identified in submarket-sized adult clam plots in two aquaculture leases in Wellfleet Harbor (Smolowitz, unpublished survey data). The seed used in these two sites had originated from a Barnstable Harbor nursery and it is strongly suspected that the pathogenic organism was transferred with the seed. Approximately 1.5 y after receiving the seed, a prevalence of 36% was identified histologically and was associated with previously noted high mortality (not quantified). Adjacent sites in the same lease and other surrounding sites did show an occasional low prevalence of disease (<8%). The two leases that had high prevalence of QPX, and high hard clam mortality, were depopulated with the help of the Wellfleet community in an attempt to halt the progression of the disease in Wellfleet Harbor. Unfortunately, approximately 2 y after that event, QPX infection and mortality became a common problem on most aquaculture sites in Wellfleet (D. Murphy personal communication). Quahog Parasite Unknown disease occurrence and severity in Wellfleet and Barnstable Harbor has decreased markedly in the last 7 y in because of a transition to oyster culture by many of the culturists. In addition, in Wellfleet, a new mortality-causing disease has occurred in the hard clam populations.
After identifying QPX as a cause of significant disease in hard clams in Massachusetts, researchers in Virginia conducted a yearlong (July 1996 to July 1997), multisample, multisite survey of wild and cultured hard clams (Ragone Calvo et al. 1998). Clams from 16 culture sites and six wild sites were sampled for QPX. Quahog Parasite Unknown was noted in two of the cultured sites and in none of the wild clams. All infected sites were located on the Atlantic side of the Eastern Shore. Prevalence ranged from 7% to 48%. The lowest prevalence was noted in July and the highest in May 1997. Mortality at that time was estimated by one aquaculturist to be 10%-20%. No swellings, nodules, or other gross lesions were noted in the affected animals.
During the summer of 2002, mortalities were noted in wild clams in Raritan Bay, NY. Histological examination of 603 clams identified QPX in 5.8% of the animals (Dove et al. 2004). Allam et al. (2004) reported that the histological prevalence in most areas of the bay was 7% or less but in some areas was 30%.
Connecticut and Rhode Island
Sunila (2006) reported a prevalence of 0.3% in wild clam surveys from the coast of Connecticut from 1997 to 2006. Quahog Parasite Unknown disease was identified once in one hard clam culture site in Rhode Island (Lyons et al. 2007). The Rhode Island aquaculturist no longer cultures hard clams on his lease and no further monitoring has occurred in that area.
MacCallum and McGladdery (2000) reported diagnostic and seasonal survey data from both hatchery and nursery and for wild clam collection locations in New Brunswick, Nova Scotia, and Prince Edward Island between 1990 and 1998. Most hatchery and nursery groups submitted were negative for QPX infection histologically, but prevalence in groups identified as infected ranged from 1.7% to a high of 80%. The higher prevalence was identified in broodstock that also exhibited morbidity. The average overall prevalence was 2.2% for all animals examined histologically. Over the same time period of examination, QPX was also found sporadically in wild animals collected as part of a seasonal survey conducted in the three locations. Histological prevalences in those samples ranged from 3.3% to 20%. At the time of the report (MacCallum & McGladdery 2000), mortality in wild clams had not been identified since the 1963 epizootic.
Gross Signs of the Disease
In Massachusetts, the parasite is noted to invade the mantle most often at the junction of the incurrent siphon with the mantle edge forming soft tan nodules or, especially in the spring, clear mucoid swellings (Fig. 2A, B). Nodules and swellings varied in size from 0.25 to 4 mm; however, small and large, sometimes irregularly shaped nodules can often be identified ventrally along the mantle edge and in the membranous mantle. Most often, the nodules are found in both the right and left mantles. Mantles of affected clams lying on the sediment surfaces were usually retracted into the shells and contained varying amounts of mucus and sand between the free edge of the mantle and the shell. The swellings and nodules are proposed to interfere with the proper functioning of the mantle. They may prevent the inner lobes of the mantle from properly forming a mantle cavity and thus interrupt normal feeding and burrowing functions. In addition, chips in the shell (and sounds noted in the initial outbreak in Provincetown, MA) were hypothesized to occur when the clam valves attempted to close on the mucus-containing sand. In a survey of cultured clams from several sites in Virginia between July 1996 and July 1997, Ragone Calvo (1998) reported a lack of macroscopically identifiable swellings or nodules in the mantle of positive animals. Similarly, swellings and nodules were not identified in infected animals in Canada (MacCallum & McGladdery 2000) or in New York (Dove et al. 2004, Dahl et al. 2010, Liu et al. 2017).
In Massachusetts, when clams were cultured in sandy soil, high numbers of infected moribund and dead animals were identified on the surface in the spring. In leases with increased clay content of the sediment (such as Duxbury, MA), empty clam shells and moribund animals were retained in the sediment (Smolowitz et al. 1998) and chipping was not noted.
DESCRIPTION OF THE QPX ORGANISM
Histologically, the organism is found in three different forms within infected tissue foci: sporangia containing mature and immature endospores and thalli (Smolowitz et al. 1998). When fully mature, sporangia, approximately 15-25 [micro]m in diameter, contain approximately 40 endospores. When the sporangia wall ruptures, the endospores, now termed thalli, are released and appear to spread in a locally invasive fashion sometimes producing a bubble-like appearance to the infections when examined histologically. The round thalli range in size from 2 to 25 [micro]m in diameter. Histologically, sporangia, thalli, and mature endospores are surrounded by either clear areas or tendrils of blue material (when examining hematoxylin and eosin tissue stains). These areas represent areas of mucus production by the parasite forming capsules around the organisms in life (mucus is washed out of the tissues when processed for histological preparations). The amount of mucus produced varies greatly, but can occupy a large halo around the thalli and sporangia and can occupy more space in the tissues than the organisms themselves.
Quahog Parasite Unknown organisms were cultured in vitro by Kleinschuster et al. (1998) from large infected nodules sampled from Massachusetts clams using a modified liquid MEM Eagle media. Microscopic examination of cultured QPX identified the three parasitic forms seen in the tissues. Although the occurrence of zoospores has been reported in culture (Whyte et al. 1994, Kleinschuster et al. 1998), it is currently thought by many that they may have resulted from contamination of the original cultures by other Labyrinthulomycotids (which are very common in the environment), especially because they have not been seen subsequently in cultured QPX organisms. Further examination of cultured QPX is needed to determine their occurrence in this species.
Transmission Electron Microscopic (TEM) Examination
Transmission electron microscopic examination of the infected clam tissue sections and samples of the cultured parasite identified characteristics of this species of Thraustochytrid (Maas et al. 1999). Thalli and immature sporangia contained electron-dense lipid bodies, mitochondria with tubular cristae, large vacuoles, and perinuclear Golgi apparatus. Interestingly, additional membrane-bound inclusions, not seen in other Thraustochytrids, and containing dark lipid material were noted in the thalli and early sporangia. More mature sporangia containing multiple nuclei that result from karyokinesis. Cytokinesis then occurs resulting in endospore production in the sporangia. Also identified as unusual for Thraustochytrids were the scale-like laminated walls of the endospores and small immature thalli. Examination of both cultured organisms and QPX-infected tissues did not identify sagenogenetosomes or ecto-plasmic nets as were described in the Canadian report (Whyte et al. 1994). Further studies of organisms cultured from hard clams in Canada (Anderson et al. 2003b) suggest that the QPX culture used for transmission electron microscopic examination in the Whyte et al. (1994) work may have been contaminated by a Labyrinthulomycota that did produce sagenogenetosomes. Although lacking sagenogenetosomes and ectoplasmic nets, many strains of QPX can produce copious mucus, a characteristic also not identified in other Labyrinthulomycota.
EFFECTS OF ENVIRONMENTAL CONDITIONS ON GROWTH OF QPX IN CULTURE
Examination of the effects of environmental conditions on the growth of QPX in culture identified parameters that both encourage and discourage growth. Brothers et al. (2000) demonstrated that the organism grows best at 24[degrees]C and dies at 32[degrees]C. No growth occurs at 0[degrees]C. Slow growth occurs as the temperature increases to 16[degrees]C. The organism grows well between salinities of 28 and 40. Mucus production is highest between a pH of 7-8, and at higher temperatures. Similar results were obtained in a later study when the effects of temperature and salinity were tested on three strains of cultured QPX (Perrigault et al. 2010). That study showed optimal growth at a salinity of 34 and inhibition of growth below a salinity of 15. Perrigault et al. (2010) noted differences in maximum growth between the Massachusetts and New York strains of QPX (20[degrees]C and 23[degrees]C, respectively).
Salinity preference was identified in the several field studies. In Massachusetts and New York, infected animals were found in areas of salinity >20 (MacCallum & McGladdery 2000, Lyons et al. 2007). Interestingly, in Virginia, in which culture of clams occurs in both high and moderate salinities, the disease only occurred in high-salinity culture sites (salinity 30-34) (Ragone-Calvo et al. 1998).
No effect of water depth has been shown to be important in the development of the disease (Lyons et al. 2007).
MOLECULAR EVALUATION OF QPX
Molecular evaluation (Maas et al. 1999) of the cultured protistian organisms placed them in the phylum Labyrinthulomycota in the family Thraustochytriidae. Further evaluation of the SSU rDNA confirmed this placement (Ragan et al. 2000, Stokes et al. 2002) and identified single-nucleotide differences between three potentially different strains. The QPX ITS regions from 12 clonal isolates were investigated (Qian et al. 2007) and found to differ slightly both between clonal isolates and even within the same clonal isolate. Interestingly, as noted by Qian et al. (2007), these findings suggest that the organisms primarily reproduce by mitosis rather than meiosis (which has been verified by culture and histological tissue evaluation). These findings also strongly suggest that within any one clonal QPX culture, genes responsible for pathogenicity may differ slightly, thus leading to differently pathogenic organisms within the same culture.
Roberts et al. (2006) first identified differentially expressed genes associated with culturing QPX at different temperatures (10[degrees]C and 21[degrees]C) and in different medias [i.e., when culturing QPX in modified MEM media (Kleinschuster et al. 1998), as compared with culturing in sterile sea water containing clam tissues]. In that study, one of the upregulated genes in the cultures at 10[degrees]C was a potassium channel protein. Later molecular examination of QPX cultures held at 10[degrees]C versus 21[degrees]C showed numerous expressed genes in culture including many proteases (Garcia-Vedrenne et al. 2013). Anderson et al. (2006) identified serine proteases in QPX extracellular proteins (ECP). In the work by Garcia-Vedrenne et al. (2013), a functional suite of genes identified in the study were those associated with regulating growth at high temperatures. Examples of genes highly expressed at 21[degrees]C were beta enolase, which can function in tissue invasion. Beta enolase may be secreted as part of the mucus and zinc metalloproteinase-disintegrins, which could result in proteases capable of degrading the extracellular matrix of host tissues. The gene Thrombospondoin-1 was identified in the transcriptome when QPX was cultured at 21[degrees]C. This gene is responsible for forming extracellular glycoprotein, which may account for the increased mucus production noted when QPX is cultured at higher temperatures (Brothers et al. 2000). Interestingly, Garcia-Vedrenne et al. (2013) found that heat shock protein 20 was actually decreased in QPX organisms cultured at 21[degrees]C and suggested that the decrease may result from rapid depletion of the transcripts at higher temperatures reflecting a more active metabolism of the QPX organisms.
Work by Perrigault and Allam (2009) identified an increased cytotoxicity of the ECP from QPX cultures grown in clam muscle tissue homogenates as compared with ECP from QPX grown in MEM. Paradoxically, however, ECP from QPX grown in MEM was more toxic to clam hemocytes than ECP from QPX cultures grown in clam muscle tissue homogenates.
Rubin et al. (2016) demonstrated that QPX organisms produce approximately 74 different extracellular peptidases, six of which are known virulence factors for pathogenic organisms, and showed that the type and abundance can vary depending on the protein substrates added to the culture media. The ECP of three stains of QPX were also examined and peptidases common to all three were a subtilisin type. Rubin et al. (2016) concluded that QPX is well adapted to a saprophytic life cycle and can produce peptidases of various types depending on the substrate it is provided.
Mucus production varies greatly by isolates (probable different strains) of QPX. Some isolates produce only moderate mucus and others produce abundant mucus in culture. In addition, some isolates lower the pH of the media as they proliferate turning the modified MEM media (Kleinschuster et al. 1998) indicator from orange/red to bright yellow (pH of 7.2 to 5.5). At least five differently behaving isolates have been cultured in Massachusetts (Smolowitz unpublished data; Gast et al. 2008). Work by Dahl et al. (2008) in which clams originating from different states were injected in a laboratory study with different isolates/strains of QPX shows that some do have different pathogenicities.
It is probable that selection of increased pathogenic genes, as identified previously, in different clones of QPX may occur within an aquaculture setting because naive animals are constantly being introduced into an infected area. This method of increasing pathogenicity in some clones of a normally opportunistic organism is new and has recently been demonstrated by Phillips and Puschendorf (2013). In their study, when exposed to large populations of naive South American frogs, a chytrid fungal infection increased significantly in virulence. It is possible that similar procedures occurred in areas such as Wellfleet and Provincetown, MA, and may account, in part, for the severity of the disease in those areas.
Confusing the pathogenicity issue for various isolates of QPX is the theory by Dahl (2008) that suggests isolates in held in laboratory culture for long periods of time may lose their pathogenic potential as occurs with in vitro culture of Perkinus marinus (Bushek et al. 1997). Although this thought is very possible, and even likely, based on the previously described work, Dahl (2008) suggested this hypothesis did not explain why his experimental results with QPX from Massachusetts, which he retrieved from ATCC, showed less pathogenicity than New York strains. That ATCC strain (deposited by Smolowitz) had been in culture for approximately 10 y before it was deposited in the ATCC so would indeed fit with Dahl's theory.
QUAHOG PARASITE UNKNOWN IN THE ENVIRONMENT
A survey of QPX occurrence in environments that also contained cultured hard clams positive for QPX disease was conducted along the eastern U.S. coast (Gast et al. 2008). Nested polymerase chain reaction (PCR) techniques (Gast et al. 2006, 2008) were used on water, sediment, seagrass, and macrophyte samples. In Virginia, samples were collected from five sites in spring, summer, and fall seasons over 2 y. Examination of clams collected from the same sites showed a low percentage of the clams from two of five sites were infected with QPX (4% and 3.3%). Only one field sample from one of these two sites was positive for QPX. Overall, a lesser number of positive environmental samples (0.66%) were detected in all environmental samples from Virginia as compared with those from Massachusetts (38.4%).
In Massachusetts, environmental samples tested from Barnstable and Pleasant Bay (where outbreaks of disease and associated mortality had occurred) were positive for QPX (38.4%), and all types of samples were positive at some point during the study (Gast et al. 2008). The highest number of positive samples were those collected from Barnstable Harbor, which was undergoing a QPX epizootic in cultured hard clams during the 2 y of the study. The highest percentage of positives were noted in seawater, sediment, and macrophyte samples in the spring season, and in seawater and sediment samples in the summer in Barnstable Harbor. In Pleasant Bay, which did not have detectable clam disease at the time of the study, the overall percentage of samples was lower, suggesting that fallowing an area may be helpful in decreasing the disease prevalence; however, an increase was noted in the fall time period in that area on the surface of snail surfaces.
Lyons et al. (2005) examined the roll of marine snow in the transmission of the diseases between animals and aquaculture farms. Marine snow (also termed flocs and marine aggregates) are mucoid aggregates of particles found in the water column that range in size from a few microns to 1 mm in diameter or length (Fowler & Knauer 1986). Using nested PCR methods (Gast et al. 2006) and in situ hybridization methods (Lyons et al. 2005), QPX organisms were detected in marine snow particles, thus confirming that marine snow is a vector of transmission and a method for spread of the organism from one infected aquaculture site to another.
Microscopic examination of clams from Massachusetts confirmed that in most cases, the mantle tissues were the first tissues infected and infection was found only at that site in many animals. As identified macroscopically, location of infection in the mantle was most often noted in the mantle tissues close to the siphon (Smolowitz et al. 1998). Importantly, Lyons et al. (2005) showed that while clams do inhale marine snow containing QPX particles into their mantle cavity, they do not ingest them and instead reject them as part of the pseudofeces. Pseudofeces are often stored at the base of the siphon until elimination from the mantle cavity. Prolonged contact by the QPX organism with the mantle at the base of the siphon most likely promotes infection of the mantle in that area (Smolowitz, personal observation; Kraeuter et al. 2011). Gill lamellae, especially the intertubular septa, are the second most common location of infection microscopically, but gill infection without mantle infection is rare (Smolowitz, personal observation; Smolowitz et al. 1998). The organism apparently gains access to the open vascular system through the mantle and spreads from there (and the gill) to vascular spaces in other parts of the body. The third most common site for proliferation of the parasite is in the vascular spaces surrounding the intestines and gonads. In metastasized infections, and rarely in initial infections, QPX is noted in vascular spaces and lumens in other areas of the body including the pericardial sac, foot, kidney, and gonadal tubule (Smolowitz et al. 1998, Dove et al. 2004).
Reports of histological tissue location vary in infected clams from states further south of Massachusetts and further north (Canada). Ragone-Calvo (1998) reported that although she did not see grossly identifiable nodules in the mantles of cultured clams from Virginia, the mantle tissue was the most common focus of infection microscopically (63%) and gill was the second most commonly infected tissue (35%). Ragone-Calvo (1998) reported that infections in clams often contained more dead and dying QPX organisms than did Massachusetts clams and the halos noted in histological sections of the tissues (representing mucus proliferation by the organisms) were smaller overall in infected tissues in clams from Virginia. Ragone-Calvo (1998) noted that mortality was not significant in areas from which clams were sampled and the prevalence of infected clams in the samples was much lower than in Massachusetts.
Dove et al. (2004) noted a prevalence of 5.8% of infected wild hard clams in animals sampled from Raritan Bay, NY. They noted potential microscopic tissue foci that may represent resolved QPX infections in some clams in which active infections were not identified. Whyte et al. (1994) reported on an outbreak of QPX in Canada in 1989. In that outbreak, organisms were identified microscopically in the connective tissue and muscle, but otherwise the histological locations of the infections were not described.
EFFECT OF SEASON IN THE DEVELOPMENT OF QPX DISEASE
After several years of experience in examining cultured hard clams with QPX disease from Barnstable, Provincetown, and Wellfleet, MA, an annual pattern of mortality was better described for that area. Clam mortality in Massachusetts most often occurred in late spring/early summer (Lyons et al. 2007). This was followed by a drop in numbers of infected surveyed and experimental animals in late summer, thought to be due to mortality of the most severely affected clams as temperatures on intertidal flats increased, stressing severely infected animals ending in mortality. Ragone-Calvo (1998) noted the highest mortalities occurred in August, October, and February in cultured clams in Virginia. In another study, Ragone-Calvo et al. (2007) noted that infection generally increased over time and mortality generally was positively associated with infections.
In New York, Liu et al. (2017) noted a seasonal pattern in two of three wild clam sites in Raritan Bay, NY, with the highest prevalences in two of the sites in August (6.6% and 13.3%) and highest prevalence in April in the third site (13.2%). A similar pattern was noted using quantitative PCR (qPCR) analysis of the tissues from the same clams. Intensities (abundance of the organism) of infection were generally low to moderate overall in these animals. Mortality was generally estimated to be moderate to low overall in all months, but was highest in April at the third site (23.3%).
It appears that prevalence in many locations appears to depend on season (MacCallum & McGladdery 2000, Allam et al. 2004, Sunila 2006, Liu et al. 2017) and probably directly reflects the local environmental effects, and the potentially increased immune capacity of the clams during increased summer temperatures (see the following paragraphs).
EFFECTS OF CLAM DENSITY ON DEVELOPMENT OF DISEASE
Drinnan and Henderson (1963) first identified QPX in a dense set of hard clams, and Dove et al. (2004) noted that the most severe infections in wild clams occurred at high-density locations in Raritan Bay, NY.
Ford et al. (2002) conducted work examining the effects of density on the spread of QPX disease in intertidal plots and subtidal experiment plots established at three clam densities (215, 430, and 860 clams/[m.sup.2]). The study demonstrated a clear effect of density on infection prevalence in the intertidal plots and a positive effect of density between the first and second prevalence samplings in the 430 clams/m subtidal plots; however, the 215 and 860 clams/[m.sup.2] plots in subtidal areas showed a consistent decrease. This experiment was initiated with animals that later were determined to have been markedly infected with QPX and were already experiencing significant mortality when the clams were moved and experimental plots were established. The actual number of infected animals planted in each plot probably varied greatly. Thus, results from the two subtidal experimental plots possibly may reflect the epidemiological changes at the end of the disease in a population, when large numbers of animals are already infected and begin to die, and not the effects of density on initial infection. Work by Dahl and Allam (2016) demonstrated that the prevalence of QPX was lower in decreased densities of clams.
Interestingly, data presented by Lyons et al. (2005, 2007) and Gast et al. (2008) demonstrate that the highest abundance of QPX in the environment occurred in areas with high percentages of severe hard clam infections (and mortality), suggesting that on death of the clam, the number of infectious organisms in the environment may increase. Increased infectious organisms in the environment could increase the infective dose of QPX filtered out of the water by a naive clam and would result in an increased number of diseased clams in the area. This helps explaining why cultured animals may be more severely affected because the continued introduction of concentrations of naive animals into an environment with high abundance of QPX organisms increases the likelihood of producing abundant infected animals that die releasing large numbers of organism and producing a high infective dose, thus perpetuating the disease. In the wild, the clams would be killed by the disease and the number of potentially new wild clams to be infected and perpetuate the disease would most likely decrease dramatically (loss of broodstock and juveniles), thus eventually decreasing the number of QPX organisms in the wild environment over time.
Based on the previously described information (environmental occurrence), and the placement of QPX in the category of an opportunistic pathogen, epidemiologically, the density of susceptible animals would be considered an important part in the development of disease.
EFFECT OF CLAM SIZE AND AGE ON DEVELOPMENT OF DISEASE
Ford et al. (1997) demonstrated that QPX was not routinely found in seed clams (primarily <16 mm in shell height); however, on examination of fifty 15-mm-shell height seeds held in an upweller on a site in Massachusetts, from which QPX-infected adults had been identified, they were positive for QPX, although at the a prevalence of 2% (Gast et al. 2006). Whyte et al. (1994) reported that infections were identified in nursery clams of 15-30 mm in shell height held in a closed-circulation quarantine system in artificial seawater; however, MacCallum and McGladdery (2000) determined that these animals were likely 1.5-2 y old and exhibited the slower growth associated with the northern distribution limits of the clam.
Quahog Parasite Unknown appeared most commonly to be identified in submarket-sized clams in culture animals in Massachusetts. Size when infection was first detected in most Massachusetts clams was between 20 and 55 mm in shell length (Lyons et al. 2007). Ragone Calvo et al. (1998) identified a range of 24-63 mm in shell height of infected clams, but animals in populations with high prevalence of the disease tended to average lower shell heights (20-45 mm).
Lyons et al. (2007) suggested that the decrease in prevalence of QPX disease in larger clams (>55 mm) may reflect an effectiveness of ingestion and digestion of QPX organisms as compared with smaller submarket-sized clams. It is also possible that larger clams are held at low densities in the aquaculture leases (clams are usually sold before reaching larger sizes), and less infective dose is available to the large clams. In addition, because wild clams tend to occur in low densities as compared with cultured clams, it is probable, based on epidemiological principles, that prevalences would be lower in wild clams in general. Lyons noted that most wild clam samples collected for evaluation were >60 mm in shell height and suggested that small-sized wild clams should be collected for evaluation in the future.
Overall, size at infection is most likely a function of dynamics that cause infection, including pathogenicity of the QPX strain, infective dose (dependent on the amount of water filtered), varying environmental temperatures, salinities, and clam density.
EFFECT OF CLAM SEX ON OCCURRENCE OF QPX
No correlation between sex and prevalence or severity of QPX disease has been demonstrated (Lyons et al. 2007; Smolowitz, unpublished data).
CONDITION INDEX (CI) OF INFECTED CLAMS
Evaluations of CI conducted on live clams from core samples (including animals on the surface as well as animals in the sediment) from infected sites in Provincetown, MA, demonstrated that the CI of infected animals was significantly lower than that of uninfected animals. Condition index data confirmed that animals infected with QPX were in poor condition when compared with uninfected animals from the same sites (18.8 versus 34.7, respectively) (Smolowitz et al. 1998). The CI was calculated as follows: CI = D[W.sub.so][W.sub.shc] x 1000.
Dahl et al. (2010) examined the CI of three strains of clams (Florida, NY notata, and New York wild) deployed at Raritan Bay. At the end of the study, there was a significantly lower CI for Florida notata strain of clam (120.61), which had experienced disease only 7 wk after deployment and had developed high morality by the end of the study (51.2%) than for the New York notata strain of clam (124.49). Condition index for both notata strains was less than the New York wild clam strain (130.24.). Condition index was calculated as follows: CI = dry soft weight (grams) x 1,000/internal shell cavity capacity (grams).
EFFECT OF CLAM STRAIN ON DEVELOPMENT OF DISEASE
Early in the investigations into the disease caused by QPX infections, culturists in Provincetown, MA, reported a lower disease severity and mortality in hatchery-spawned seed clams produced from local Massachusetts broodstock versus seed clams imported from New Jersey or farther south along the U.S. coast line. This was also observed in preliminary data from a Provincetown-based experiment that was disrupted before being completed (Smolowitz, unpublished data). Ford et al. (2002) identified a difference in occurrence of disease in the seed acquired from South Carolina and planted in New Jersey. Clams of that origin developed higher prevalence and mortality than the New Jersey seed planted in New Jersey.
Ragone-Calvo et al. (2007) identified a gradation in disease prevalence and associated mortality that directly correlated with origin of the seed and location of the aquaculture site in a garden variety, 2-y field study. The study demonstrated that seeds spawned from northern clam stocks (Massachusetts and New Jersey) planted in both locations (New Jersey and Virginia) had lesser prevalence of QPX disease, and lower cumulative mortality over the 2.5 y of the study than did seed stocks spawned from southern-origin broodstock from Florida and South Carolina that were also planted in New Jersey and Virginia locations. In addition, VA-originated seed clams planted in Virginia had lower disease prevalence and mortality in Virginia field sites than those observed in New Jersey for the Virginia-origin seeds.
Ragone-Calvo et al. (2007) hypothesized that the innate immune system of southern-origin stocks that have been selected over generations for the ability to survive in warmer climates may not be able to adapt to the annual local environmental changes of more northern locations and local stocks could, thus increasing the potential for QPX infection, especially in the spring and fall when the innate immune systems of southern clams might be more severely suppressed by cooler environmental temperatures. This was further supported by the occurrence of high mortality in southern seeds initially deployed at the sites at the beginning of the study (Ragone-Calvo et al. 2007). This initial mortality, occurring in the first spring after deployment, was not due to QPX or other known diseases in the Florida and New Jersey seed planted in New Jersey and Virginia, and it was suggested that it resulted from poor adaption of the southern seed to northern climates.
Kraeuter et al. (2011) followed the prevalence of QPX disease and mortality in large seeds originating from South Carolina, NJ, and Massachusetts over the course of 1.5 y (spring 2008 to fall 2009). Infection severity and prevalence was higher in clams originating in South Carolina than in Massachusetts or New Jersey-origin clams in both locations. Interestingly, there was higher survival in all three strains of clams planted in New Jersey than in Massachusetts. The authors suggested that this could be due to higher QPX pressure (high prevalence of infected clams and infectious QPX organisms in the bay) on clams in the Massachusetts location or higher stress due to location in the intertidal area of the Massachusetts locations (versus subtidal in New Jersey).
Dahl et al. (2010) conducted a study in which seeds from Florida were compared with seeds from New York notata broodstock and New York first generation wild broodstock. Animals were placed in four areas within New York estuaries. Quahog Parasite Unknown infections, disease, and mortality were noted only in Raritan Bay. Disease severity and clam mortality was measured semiannually over 2 y. Disease prevalence and cumulative mortality for animals deployed in Raritan Bay were the highest in Florida clams at the end of the 1.5 y of the study (51.2% and 68.0%, respectively); disease severity was low in New York notata seeds (2.5%), but mortality was high (59.0%) and infection was not identified in seeds originating from local wild clams [although cumulative mortality (68.9%) was high in this group also]. This work added further confirmation that southern seeds planted in northern areas exhibited more severe disease, but the study was not able to associate disease with higher mortalities.
Dahl et al. (2008) conducted a laboratory study in which seed clams produced from broodstock collected from hatcheries in Massachusetts, NY, Virginia and Florida were injected in the pericardial sac with three strains of QPX and were held at 21[degrees]C-22[degrees]C. The work confirmed that seeds spawned from southern broodstock developed more severe disease and experienced higher associated mortality even at the northern summertime temperatures. Interestingly, the normal summertime temperature of culture sites in Florida (https://www.nodc.noaa.gov/dsdt/cwtg/egof.html) is consistently higher than that of New York (https://www.nodc.noaa.gov/dsdt/cwtg/catl.html). This may have resulted in innate immune responses in Florida clams that may not function as effectively as at the "cooler" summer temperature of New York.
The studies conducted to date point to the susceptible of Florida clam strains cultured in more northern areas and confirmed that there are probably genetic differences in the innate immune systems between clam strains that may result in differences of expression at lower temperatures.
In areas of potentially high prevalence and disease severity, QPX infection in a population progresses through the population over the course of 1.5-2 y (depending on season deployed and various other factors discussed here). Epidemiologically, and as discussed previously, a gradual increase in number of clams infected is balanced by the number that die and the density of the remaining uninfected animals resulting in a misleading decrease in the percent infected (Fig. 3). This information highlights the need for continuous monitoring to enable the determination of trends. Point in time prevalence does not provide a complete picture of the disease progression in a population.
THE INNATE IMMUNE RESPONSE OF CLAMS TO THE QPX ORGANISM
The swellings and nodules in the mantle and other areas of infection in the clams seen microscopically during the spring and summer in most coastal locations are all similar in appearance histologically.
The QPX organisms usually incite a strong immune response by the clam hemocytes in the warmer temperature of the summer (Fig. 4A). Histologically, the inflammation (Ragone-Calvo et al. 1998, Smolowitz et al. 1998) consist of an abundant admixture of both granular and agranular hemocytes (thousands of cells). Hemocytes migrating toward the infecting parasite seem to vary in the ability to move through the thick mucus coat surrounding the proliferating organisms. This results in variable phagocytosis of the QPX organisms (Ragone-Calvo et al. 1998, Smolowitz et al. 1998, Anderson et al. 2003a). During the summer, phagocytosis of the QPX organism appears to occur to a high degree overall, with a decrease in the mucus halos and accumulation of large irregular, stippled to homogenous, basophilc to eosinophilc (hematoxylin and eosin tissue stain), often round, inclusions identified in the phagocytic hemocytes. These inclusions appear in many cases to be dead thalli, sporangia, and/or endospores. Multinucleated giant cells, a rarely noted inflammatory cell in bivalve inflammation, are commonly noted in the summer, especially in severely QPX-infected clam tissues and appear to be an important part of the phagocytic response in hard clams (Fig. 4B). The multifocally extensive areas of hemocytic response to the infecting parasites result in firm, tan swellings, and nodules in the mantle seen macroscopically in Massachusetts in the summer time period.
Interestingly, in Massachusetts, this author has noticed a difference in the macroscopic characteristics of the swellings and nodules and in the histological appearance of the inflammatory response in the spring versus summer. In early spring in Massachusetts (April to May), a higher abundance of clear, mucoid swellings/nodules (as compared with firm tan nodules) are seen macroscopically, which correlated microscopically with infecting QPX organisms surrounded by abundant mucus in the mantles and other tissues (Fig. 5A). At these times and in these animals, the hemocytic inflammatory response is often markedly less that that seen in the summertime and is instead characterized by low numbers of hemocytes around the areas of QPX infection (Fig. 5B). In the fall time period, often both types of infections/immune reactions are noted in the tissues histologically (Fig. 2B) (NRAC Annual Report, July 1, 2006 through August 31, 2008). These histological analyses seem to confirm a seasonal or temperature vulnerability in the clam innate immune response.
Evaluation of the innate immune response has been examined using flow cytometry to assess hemocytes freshly collected from juvenile uninfected, cultured clams from Massachusetts (NRAC Annual Report, July 1, 2006 through August 31, 2008). Briefly, that work demonstrated an increase in the number of circulating hemocytes, phagocytosis ability, and production of reactive oxygen species in the summer months versus the spring, indicating a more active summertime innate immune system in the clams. Work by Perrigault et al. (2011) described a laboratory experiment that accessed changes in defensive parameters of Florida-origin infected and uninfected clams versus infected clams from Massachusetts at three temperatures: 13[degrees]C, 21[degrees]C, and 27[degrees]C. Findings in that study varied by treatment and length of time maintained in laboratory tanks, but it was concluded that at 21[degrees]C, there was an increase in the total circulating hemocytes (TCH) in uninfected Florida-origin clams versus 13[degrees]C and 27[degrees]C; however, in clams originating from Massachusetts, the TCH was lower at 21[degrees]C. This finding is contrary to previous work in bivalves that identified TCH counts in bivalves held at higher temperatures. Histologically, there are large areas of inflammation in the tissues associated with QPX at 21[degrees]C, suggesting to this author (Smolowitz) that the THC is lowered at this temperature in infected Massachusetts clams because of sequestering of hemocytes at the infection site. A similar decrease in circulating inflammatory cell abundance (leukopenia) is a common finding in severe vertebrate infections (Duncan & Prasse 1986).
Perrigault et al. (2011) demonstrated significant differences in the immune response of Florida-origin clams at 13[degrees]C versus 21[degrees]C; specifically, in the resistance of hemocytes to cytotoxicity by QPX ECP and by induction of anti-QPX activity in the plasma. Similar, and stronger, anti-QPX activities were noted in clams from Massachusetts infected with QPX, indicating a difference between these two strains of clam.
Wang et al. (2016a) identified higher levels of four heat shock proteins in clams maintained at variable air temperatures higher than 18[degrees]C for exposure times ranging from 2 to 18 h at one day after treatment but detected an effect of decreased QPX prevalence in only animals held at for 2 h at 27[degrees]C. Mortality increased with increasing temperature and mortality was 8% at this treatment level.
LABORATORY INFECTION METHODS
Laboratory-based infections provide a means to study disease in more controlled conditions than in field research. Smolowitz et al. (2001) designed an experiment in which the naive seed was exposed to QPX in a flow through raceways. In that study, animals were either exposed to cultured QPX in the water column or to potentially infected clams from Province-town, MA (an area that was identified in previous studies as being heavily infected), or clams were injected in the pericardial sac with cultured QPX. Treatments were conducted in duplicate, and duplicate control tanks were also established. Animals were held in carboys and filtered, and flow-through sea water was heated to two different temperatures. The temperature was not set but was allowed to vary with the incoming sea water temperature over the course of the experiment (one temperature was maintained at that of the incoming seawater and the other was heated to approximately 2[degrees]C-4[degrees]C above the incoming sea water). Animals were held in the raceways for 2 y and sampled three times during the project (after the initial deployment examination). No clams were positive for QPX at any time in the injected clams and water column exposed animals at any sample period; however, naive clams exposed to infected clams were positive for the disease at the first sample period and severities of infection and mortality increased markedly as the experiment continued. By the end of the second year, mortality in both of the groups (high and low temperature) had reached 55% (compared with 18% and 8% in control groups, which were not infected with QPX). The highest prevalence of infection (69% of all animals sampled) occurred in the spring sample in animals held at the lower temperature (Smolowitz et al. 2001; Smolowitz, unpublished data) (Fig. 6).
Dahl and Allam (2007) conducted a similar laboratory-based study. All attempts to transfer the disease occurred in clams that were held in recirculating tanks at 10[degrees]C-21[degrees]C and fed daily with algal concentrate. In the first experiment, naive seed clams were cohabitated with infected adult clams from Barnstable Harbor, MA (another focus of severe disease). Quahog Parasite Unknown disease did not develop in the seed clams after 10 mo of cohabitation. It is possible that they were not successful in causing the disease because of the lack of temperature change in the experimental design.
In another experiment, Dahl and Allam (2007) exposed adult naive clams to cultured QPX in three ways: by adding cultured QPX to the water column, by direct application of QPX culture into the open mantle cavity while feeding (palladial application), and by injection of the organism into the pericardial sac at a concentration of 4 x [10.sup.4] QPX cells/clam. Palladial application of QPX resulted in infection in 2 of 11 animals examined. High prevalence was noted in injected animals at 2 wk (83%), 14 wk (42%), and at 31 wk (43%) after injection and the infections progressed in clams over the time period of the experiment. Mortality in the injected clam group was 45% by 16 wk and 76% by the end of the trial. A second experiment was conducted in which naive seed clams were injected in the pericardial sac with 6 x [10.sup.3] QPX cells/clam. Similar results occurred in this experiment.
The direct association of seed clams with infected adults, combined with a temperature change of incoming water, indicates that the variation in temperature from low to high may be as important as the temperature of the water in the pathogenesis of the disease in naive clams. As the innate immune system "gears up," its ability to fight off QPX may lag behind the ability of the QPX organism to invade the mantle tissues. Direct infection experiments could provide information about how infection first occurs. The development of the pericardial injection method provides an important and easily usable laboratory infection model to identify progression of the disease, but it does not provide information about how initial infection occurs.
The diagnosis of QPX can be accomplished in several ways. On shucking, the shucker should always carefully examine the mantle for swellings and nodules along the edges, especially the area at the base of the siphon. An easy determination of QPX infection can be made by removing the piece of potentially infected mantle tissue, cutting into the nodule or swelling, and touching the cut surface of the nodule to a microscope the slide. This "touch preparation" can be coverslipped and examined immediately on a microscope or dried and stained with a quick stain such as Protocol (Fisher Scientific) for later microscopic observation.
Histological evaluation of tissues should include the traditional sectioning through the body of the clam (Howard et al. 2004), but additional tissue sections should also be examined. It is advantageous to include a section through the heart and kidney because infections often can be found at the dorsal extent of the gills in that area and in the dorsal water tubule. Importantly, a wedge of mantle collected from the base of the siphon should be included. If nodules and swelling are noted macroscopically in the mantle tissue, these should be included in the sections. Paraffin embedding and subsequent 5-um sectioning with hematoxylin and eosin staining provides appropriate diagnostic histological sections. Because the QPX organisms are relatively large, compared with the surrounding hemocytes and other cells, histological detection is easy. Ragone-Calvo (1998) published a method for evaluating the severity of QPX infection in histological tissue sections as follows: rare (1-10 cells), light (11-100 cells), moderate (101-1,000 cells), and heavy (> 1,000 cells).
Special stains have been used in the past on histological sections. Aqueous Periodic acid--Schiff stains the spore walls and cytoplasm of the thalli but not the nucleus or the surrounding mucus (if any remains in the section) (Whyte et al. 1994, Smolowitz et al. 1998). Grocotts methenamine sliver stain gave mixed results in different laboratories but did stain the organism. It strongly stained the thalli nuclei in one study (Smolowitz et al. 1998) and the cell wall in another (Whyte et al. 1994).
Molecular methods of identification were first developed by Stokes et al. (2002). Polymerase chain reaction primers to portions of the SSU rDNA (QPX-F and QPX-R2) were developed and field tested for sensitivity and specificity for QPX. When compared with histological examination, the PCR method was not as sensitive (less animals were identified as positive via PCR than histology). Reamplification of the initial product resulted in increased sensitivity but still did not equal histological results. Importantly, false negatives occurred and were thought to result primarily from sampling of animals with small foci of infection when both histological and PCR samples were removed from the same animal. Unlike many other bivalve diseases, QPX is not widespread in the vascular system in early to midstages of infection, so tissue sampling can easily cause false negatives. Polymerase chain reaction did detect animals positive for QPX that were negative by histological examination, again indicating tissue selection problems; however, it is possible that QPX organisms may also have been present on the mantle cavity surface of the mantle and had not yet invaded the tissues. Evidence for this possibility is noted in work by Gast et al. (2006) in which two groups of 15-mm clams from a nursery from an area, in which infected adult clams were found, were examined for QPX. In that study, one of 50 clams was positive histologically for QPX in group A and none in group B, but using DGGE methods (see the following paragraphs) on 50 animals from each group, 12% of group A and 10% of group B were positive. It is hypothesized that many of the QPX organisms were attached to the tissue surfaces only and had not yet invaded the animals.
Stokes et al. (2002) developed sensitive and specific in situ hybridization methods using a cocktail of two probes to QPX SSU rDNA (QPX 641 and QPX 1318). The combination of these two probes provided excellent staining of the parasites in tissue sections. In situ hybridization staining can be used on touch preparations (discussed previously) to positively identify QPX organisms.
A DGGE-based detection method was developed for use with seawater/marine snow, sediment and surface scraping of macrophytes and sea grass, and for clam tissue for identification and quantification of QPX (Gast et al. 2006). Results demonstrated that the method could detect levels as low as 10 cells/200 mL of seawater or 10 cells/gram of sediment. Nested PCR methods combined with DGGE methods can provide separation of fragments with only a single base difference resulting in a method for potentially determining strain differences and identifying environmental QPX occurrence.
Lyons et al. (2006) developed a sensitive and specific SYBR Green-based, qPCR test method using primers (qpxIRKl and qpx IRK2) that amplified a 312-base pair product of a targeted transcript (GenBank accession number DV942140), which enhances the specificity of the method versus using ribosomal DNA. Using cultured QPX dilutions, the method was sensitive down to one-fifth of a single organism. It is important to note that in culture (and in tissue samples), the number of nuclei varies greatly depending on the stage of development from thalli, which has one copy of the DNA, to sporangia that can contain 40 copies of DNA in maturing endospores, thus resulting in the ability to detect less than one organism on average. Specificity was determined by testing the method with 27 different closely related Labyrinthulomycetes (determined by previous identification using general Labyrinthulomycete primers). The method was effective in determining the occurrence and abundance of QPX in marine snow and pseudofeces and in clam tissues. Importantly, the method did not detect the QPX infection in nodules from clams with nonviable organisms as determined histologically.
Liu et al. (2009) developed a qPCR method based on the 5.8S rRNA and internal transcribed spacer (ITS) genes of QPX (QPX 5.8S rDNA and QPX-ITS2-R2). Specificity was determined by comparing the primers with three other Thraustochytrid available from American Type Culture Collection (Manassas, VA) and by comparing sequences available in GenBank for three labyrinthulids and 30 other more distantly related species of heterokonts. Sensitivity was determined by developing plasmids containing the ITS region of interest and equating the average number of ITS regions to cycle threshold counts in a cell. As in the qPCR reaction described previously (Lyons et al. 2006), the average number of ITS regions per cell was based on the cultured QPX cells, which is highly variable. Liu et al., determined that their method was able to detect 0.05 QPX cells per reaction.
The method developed by Liu et al. (2009) was used to detect QPX in sediment and water column samples from Raritan Bay, NY. Great variation in results based on the methods of handling and extraction was noted. It was also used to quantify QPX infection in clam tissues and the results were compared with histological evaluations of the same clams. In their work, no false negatives (animals negative by qPCR but positive by histology) were identified. Liu et al. (2009) sampled whole mantle homogenate instead of small pieces of mantle. This method may be better suited to localized QPX infections that may not be seen macroscopically and/or have not spread throughout the tissue. Whereas histology does not detect all infections (thus resulting in false negatives), whole mantle sampling may potentially provide false positives because qPCR methods conducted on mantle tissue may identify QPX attached to the mantle surface, but not invading the tissues, thus showing exposure but not necessarily infection as demonstrated in the Gast et al. (2006) study.
Wang et al. (2016b) showed that holding infected clams at 27[degrees]C for 2 h significantly reduced the prevalence of QPX infection in the research samples and suggested that a heating procedure might be incorporated into culture situations.
Work by Dahl and Allam (2016) identified a lower occurrence of QPX in hard clams maintained during the summer in higher temperature/lower salinity environments and suggested relocation within the same estuary/bay as a method of disease mitigation if possible. There is danger, however, in relocation if the new area does not have substantially higher temperatures and/or lower salinities than the original infected area because a potentially pathogenic isolate of QPX and QPX abundance in the environment may be increased in the new area with movement of infected clams.
Letting effected areas lie fallow for an unknown period of time may work but has not been shown to help significantly at this point in time, and because of the nature of aquaculture in the United States, most culturists do not have multiple sites to use or not use. Decreasing density of clams in the environment is helpful. Using strains of clams acclimated to the aquaculture environment is important. As with other bivalve diseases, the best hope for reducing the effects of this disease in cultured animals is development of resistant/tolerant clams.
Recent work using proteomic methods is being used to identify genetic markers that may identify clams with increased ability to survive or evade severe disease. This work will identify markers for resistant/tolerant broodstock for use in aquaculture (NRAC Annual Report September 1, 2015 to August 30, 2016).
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ROXANNA SMOLOWITZ (*)
Aquatic Diagnostic Laboratory, Roger Williams University, One Old Ferry Road, Bristol, RI 02809
(*) Corresponding author. E-mail: firstname.lastname@example.org
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|Publication:||Journal of Shellfish Research|
|Date:||Oct 1, 2018|
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