Smolowitz Roxanna M.

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Smolowitz

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Roxanna M.

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Environmental distribution and persistence of Quahog Parasite Unknown (QPX)

(Inter-Research, 2008-09-24) Gast, Rebecca J. ; Moran, Dawn M. ; Audemard, Corinne ; Lyons, M. Maille ; DeFaveri, Jacquelin ; Reece, Kimberly S. ; Leavitt, Dale F. ; Smolowitz, Roxanna M.

Quahog Parasite Unknown (QPX) is the cause of mass mortality events of hard clams Mercenaria mercenaria from Virginia, USA, to New Brunswick, Canada. Aquaculture areas in Massachusetts, USA, have been particularly hard hit. The parasite has been shown to be a directly infective organism, but it is unclear whether it could exist or persist outside of its clam host. We used molecular methods to examine water, sediment, seaweeds, seagrass and various invertebrates for the presence of QPX. Sites in Virginia and Massachusetts were selected based upon the incidence of QPX-induced clam die-offs, and they were monitored seasonally. QPX was detectable in almost all of our different sample types from Massachusetts, indicating that the parasite was widely distributed in the environment. Significantly more samples from Massachusetts were positive than from Virginia, and there was a seasonal pattern to the types of samples positive from Massachusetts. The data suggest that, although it may be difficult to completely eradicate QPX from the environment, it may be possible to keep the incidence of disease under control through good plot husbandry and the removal of infected and dying clams.
Chemical impacts in fish and shellfish from Cape Cod and Massachusetts Bays

(Barnstable County Department of Health and Environment, 1998) Moore, Michael J. ; Smolowitz, Roxanna M. ; Leavitt, Dale F. ; Stegeman, John J.

Mununichogs, soft shell clams, and blue mussels from some or all of 10 sites in Boston Harbor and Massachusetts and Cape Cod Bays were examined histologically: a suite of pathological changes previously known to be associated with chemical contamination were found in animals from the more contaminated sites. In particular, liver tumors were evident in 14% of the adult mununichogs from the Island End River, a tributary of the Mystic River in Boston Harbor. Additionally, a number of pathologies previously shown to be associated with chemical exposure were seen in the two bivalve species at a number of contaminated sites. Induction of cytochrome P45() IA (CYPIA) was also seen in muntntichogs from the more contaminated sites: CYPIA induction is a biochemical change associated with exposure to dioxin and other planar halogenated and aromatic hydrocarbons. These findings suggest that there are measurable biochemical and pathological changes in intertidal fish and shellfish from the more contaminated parts of the Massachusetts Bays system. These types of changes were less evident in the two reference sites in Cape Cod Bay.
Lethal marine snow : pathogen of bivalve mollusc concealed in marine aggregates

( 2005-06-14) Lyons, M. Maille ; Ward, J. Evan ; Smolowitz, Roxanna M. ; Uhlinger, Kevin R. ; Gast, Rebecca J.

We evaluated marine aggregates as environmental reservoirs for a thraustochytrid pathogen, Quahog Parasite Unknown (QPX), of the northern quahog or hard clam, Mercenaria mercenaria. Positive results from in situ hybridization and denaturing gradient gel electrophoresis confirm the presence of QPX in marine aggregates collected from coastal embayments in Cape Cod, Massachusetts, where QPX outbreaks have occurred. In laboratory experiments, aggregates were observed and recorded by entering a quahog’s pallial cavity, thereby delivering embedded particles from the water column to its benthic bivalve host. The occurrence of pathogen-laden aggregates in coastal areas experiencing repeated disease outbreaks suggests a means for the spread and survival of pathogens between epidemics and provides a specific target for environmental monitoring of those pathogens.
Epizootiology of Quahog Parasite Unknown (QPX) disease in northern quahogs (=hard clams) Mercenaria mercenaria

(National Shellfisheries Association, 2007-08) Lyons, M. Maille ; Smolowitz, Roxanna M. ; Gomez-Chiarri, Marta ; Ward, J. Evan

The economically important marine bivalve mollusc, Mercenaria mercenaria, (commonly called a northern quahog or hard clam), has endured considerable mortalities caused by a thraustochytrid pathogen called Quahog Parasite X (QPX). Data on the percent prevalence of QPX infections were compiled from published reports along with our data to describe the epizootiology of QPX disease. QPX infections occurred in clams collected from both cultured beds and wild populations, but a higher percentage of QPX cases (76.5%) were from cultured clam beds. In addition, samples from cultured beds had a significantly higher prevalence (29.2 ± 27.2%) of QPX infections compared with samples from wild populations (9.6 ± 9.6%). The highest prevalence of QPX infections occurred in clams from samples with an intermediate size range (shell lengths 20–55 mm). QPX infections occurred in both male and female clams, but infection prevalence does not appear to be correlated with sex or sex ratios. The geographical range of QPX-related clam mortalities was Atlantic Canada to the Eastern Shore of Virginia, USA. Only marginally significant differences were detected between the prevalence of QPX at different locations. There were no latitudinal gradients in QPX prevalence or frequencies, suggesting local factors were important in determining its distribution. Although QPX infections occurred throughout the year, no seasonal trends in the prevalence or frequencies of QPX were discernable. This summary of information available on QPX disease highlights the need for more thorough data collection regarding factors believed to be associated with its presence and severity in hard clams.
Development of a real time quantitative PCR assay for the hard clam pathogen Quahog Parasite Unknown (QPX)

(Inter-Research, 2006-09-14) Lyons, M. Maille ; Smolowitz, Roxanna M. ; Dungan, Christopher F. ; Roberts, Steven B.

Quahog Parasite Unknown (QPX) is a thraustochytrid pathogen responsible for catastrophic mortalities of the northern quahog (hard clam) Mercenaria mercenaria. A real-time quantitative polymerase chain reaction (qPCR) assay was developed to assist research efforts on QPX ecology and pathology. Sensitivity of the assay was evaluated with serial dilutions of QPX-cultured cells to determine the lowest concentration of DNA that remained detectable in both the presence and absence of extraneous environmental substances. QPX cells were quantified before DNA extraction to calibrate standard curves to cell counts. Based on our results, the qPCR assay is able to quantify QPX within the range of 1 to several thousand organisms per reaction. Specificity of the assay was assessed by testing 29 thraustochytrid-like protists isolated from suspension-feeding bivalves from China, Oregon, Maryland, and Virginia. Application of the assay was demonstrated with positive qPCR results from naturally contaminated environmental samples including marine aggregates (i.e. marine snow), clam pseudofeces, and inflammatory nodules from infected clams. This quantitative assay for QPX will provide a valuable tool for characterizing QPX parasite abundances in coastal environments and for improving clam disease diagnostics.
Culture-dependent characterization of the microbial community associated with epizootic shell disease lesions in American lobster, Homarus americanus

(National Shellfisheries Association, 2005-10-01) Chistoserdov, Andrei Y. ; Smolowitz, Roxanna M. ; Mirasol, Feliza ; Hsu, Andrea

Epizootic shell disease in the American lobster is an important factor affecting lobster fisheries in and around the Long Island Sound. It is a strictly dermal disease, because no correlation was observed between occurrence of epizootic shell disease and hemolymph infection. The culturability of bacteria from lesions was variable and averaged around 1.1%. The lesions contained two to four orders of magnitude more bacteria than healthy carapace surfaces of the same animal. Chitinoclastic bacteria comprised a very small fraction of bacteria present in the lesions, suggesting that their role in epizootic shell disease may be limited. Phylogenetic analysis of bacteria isolated from the lesions showed no typical bacterial pathogens of lobsters such as Aerococcus viridans or Vibrio fluvialis. Moreover, bacteria commonly associated with shell disease of other Crustacea or other forms of shell disease of the American lobster were not found. Two common groups of bacteria were isolated from lesions of all lobsters used in this research: one belonging to a species complex affiliated with the Flavobacteriaceae family and the second belonging to a series of closely related if not identical strains of Pseudoalteromonas gracilis. Bacteria isolated from only a few lobsters were related to Shewanella frigidimarina, Alteromonas arctica, Vibrio lentus, Shewanella fidelia, Pseudoalteromonas tunicata and Vibrio spp. Based on the analyses of culturable isolates, overall microbial communities found in lesions of lobsters from eastern Long Island Sound and Buzzards Bay appear to be similar to each other.
DGGE-based detection method for Quahog Parasite Unknown (QPX)

(Inter-Research, 2006-06-12) Gast, Rebecca J. ; Cushman, E. ; Moran, Dawn M. ; Uhlinger, Kevin R. ; Leavitt, Dale F. ; Smolowitz, Roxanna M.

Quahog Parasite Unknown (QPX) is a significant cause of hard clam Mercenaria mercenaria mortality along the northeast coast of the United States. It infects both wild and cultured clams, often annually in plots that are heavily farmed. Subclinically infected clams can be identified by histological examination of the mantle tissue, but there is currently no method available to monitor the presence of QPX in the environment. Here, we report on a polymerase chain reaction (PCR)-based method that will facilitate the detection of QPX in natural samples and seed clams. With our method, between 10 and 100 QPX cells can be detected in 1 l of water, 1 g of sediment and 100 mg of clam tissue. Denaturing gradient gel electrophoresis (DGGE) is used to establish whether the PCR products are the same as those in the control QPX culture. We used the method to screen 100 seed clams of 15 mm, and found that 10 to 12% of the clams were positive for the presence of the QPX organism. This method represents a reliable and sensitive procedure for screening both environmental samples and potentially contaminated small clams.
Development and validation of a real-time quantitative PCR assay for the detection and quantification of Perkinsus marinus in the Eastern oyster, Crassostrea virginica

(National Shellfisheries Association, 2009-08) DeFaveri, Jacquelin ; Smolowitz, Roxanna M. ; Roberts, Steven B.

Perkinus marinus causes a devastating disease, known as Dermo, in the Eastern oyster Crassostrea virginica. Routine detection of the disease is traditionally accomplished by the use of the Ray/Makin assay, using Fluid Thioglycollate Medium (RFTM). A simple real-time quantitative PCR assay was developed as a diagnostic tool to detect and quantify P. marinus, to complement and serve as an alternate to the RFTM method. Using a dual-labeled probe approach, a sensitive assay was designed to accurately detect a range of one to several thousand P. marinus organisms present in oyster tissues. A simple extraction method was used to increase throughput of the assay. Cultured P. marinus cells were quantified prior to DNA extraction, generating a standard curve and allowing cell counts to be derived from PCR cycle threshold values. Direct comparison of the RFTM and real-time PCR methods was accomplished by using tissue samples from the same oyster for both tests. Plotting cycle threshold values against the known Mackin index value generated a standard curve with a coefficient of regression of 0.9. Our results indicate that correlations could be made between this molecular based approach and traditional methods, allowing results generated from the PCR assay to be easily translated into the understood Mackin scale.
Developmental expression of the Nfe2-related factor (Nrf) transcription factor family in the zebrafish, Danio rerio

(Public Library of Science, 2013-10-24) Williams, Larissa M. ; Timme-Laragy, Alicia R. ; Goldstone, Jared V. ; McArthur, Andrew G. ; Stegeman, John J. ; Smolowitz, Roxanna M. ; Hahn, Mark E.

Transcription factors in the CNC-bZIP family (NFE2, NRF1, NRF2 and NRF3) regulate genes with a wide range of functions in response to both physiological and exogenous signals, including those indicating changes in cellular redox status. Given their role in helping to maintain cellular homeostasis, it is imperative to understand the expression, regulation, and function of CNC-bZIP genes during embryonic development. We explored the expression and function of six nrf genes (nfe2, nrf1a, nrf1b, nrf2a, nrf2b, and nrf3) using zebrafish embryos as a model system. Analysis by microarray and quantitative RT-PCR showed that genes in the nrf family were expressed throughout development from oocytes to larvae. The spatial expression of nrf3 suggested a role in regulating the development of the brain, brachia and pectoral fins. Knock-down by morpholino anti-sense oligonucleotides suggested that none of the genes were necessary for embryonic viability, but nfe2 was required for proper cellular organization in the pneumatic duct and subsequent swim bladder function, as well as for proper formation of the otic vesicles. nrf genes were induced by the oxidant tert-butylhydroperoxide, and some of this response was regulated through family members Nrf2a and Nrf2b. Our results provide a foundation for understanding the role of nrf genes in normal development and in regulating the response to oxidative stress in vertebrate embryos.
Host susceptibility hypothesis for shell disease in American lobsters

(American Fisheries Society, 2007-12-31) Tlusty, Michael F. ; Smolowitz, Roxanna M. ; Halvorson, Harlyn O. ; DeVito, Simone E.

Epizootic shell disease (ESD) in American lobsters Homarus americanus is the bacterial degradation of the carapace resulting in extensive irregular, deep erosions. The disease is having a major impact on the health and mortality of some American lobster populations, and its effects are being transferred to the economics of the fishery. While the onset and progression of ESD in American lobsters is undoubtedly multifactorial, there is little understanding of the direct causality of this disease. The host susceptibility hypothesis developed here states that although numerous environmental and pathological factors may vary around a lobster, it is eventually the lobster's internal state that is permissive to or shields it from the final onset of the diseased state. To support the host susceptibility hypothesis, we conceptualized a model of shell disease onset and severity to allow further research on shell disease to progress from a structured model. The model states that shell disease onset will occur when the net cuticle degradation (bacterial degradation, decrease of host immune response to bacteria, natural wear, and resorption) is greater than the net deposition (growth, maintenance, and inflammatory response) of the shell. Furthermore, lesion severity depends on the extent to which cuticle degradation exceeds deposition. This model is consistent with natural observations of shell disease in American lobster.
Influence of host genetic origin and geographic location on QPX disease in northern quahogs (=hard clams), Mercenaria mercenaria

(National Shellfisheries Association, 2007-04) Ragone Calvo, Lisa M. ; Ford, Susan E. ; Kraeuter, John N. ; Leavitt, Dale F. ; Smolowitz, Roxanna M. ; Burreson, Eugene M.

QPX (Quahog Parasite Unknown) a protistan pathogen of northern quahogs (=hard clams), Mercenaria mercenaria, has caused disease outbreaks in maritime Canada, and in Massachusetts, New York, New Jersey, and Virginia, USA. Although epizootics have occurred in wild hard clam populations, the parasite has most seriously affected cultured hard clams, suggesting that aquaculture practices may promote or predispose clams to the disease. In this investigation the influence of clam genetic origin and the geographic location at where they are grown on QPX disease susceptibility was examined in a common garden experiment. Aquaculture stocks were acquired from hatcheries in Massachusetts, New Jersey, Virginia, South Carolina, and Florida and spawned at a single hatchery in Virginia. All stocks were originally, although not exclusively, derived from wild hard clam populations from each state. The seed clams were deployed at two sites, New Jersey and Virginia, and evaluated during the subsequent 2.5 y for growth, survival, and QPX disease. At both sites, South Carolina- and Florida-derived clam stocks exhibited significantly higher QPX prevalence and lower survival than New Jersey and Massachusetts clam stocks. Levels in the Virginia stock were intermediate. In Virginia, mortality at the termination of the experiment was 78%, 52%, 36%, 33%, and 20% in the Florida, South Carolina, Virginia, Massachusetts, and New Jersey hard clam stocks, respectively. Mortality was significantly correlated with QPX prevalence. Maximum QPX prevalence in the South Carolina and Florida stocks ranged from 19% to 21% and 27% to 29%, respectively, whereas in the Virginia, New Jersey, and Massachusetts stocks prevalence was 10% or less. Similar trends were observed in New Jersey where mortality at the termination of the experiment was estimated to be 53%, 40%, 20%, 6%, and 4% in the Florida, South Carolina, Virginia, Massachusetts, and New Jersey clam stocks, respectively. QPX prevalence peaked at 18% in the Florida stock, 38% in the South Carolina, 18% in the Virginia, and 5% in the New Jersey and Massachusetts stocks. These results suggest that host genotype is an important determinant in susceptibility to QPX disease. As such, hard clam culturist should consider the genetic origin of clam seed stocks an important component of their QPX disease avoidance/management strategies.

Smolowitz Roxanna M.

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