Immunological assays of hemocytes in the Northern Quahog Mercenaria mercenaria.

The northern quahog Mercenaria mercenaria (commonly named hard clam) is an important aquaculture and fishery species along the Atlantic west coast. Environmental stresses, such as heat shock, fluctuating salinity, and harmful algal blooms are major challenges for clam aquaculture. In response to environmental stresses, hemocytes would change dynamically for defense and immunity. The goal of this study was to characterize basic immunological assays of hemocytes in the northern quahog by use of flow cytometry. The objectives were to: 1) develop a non-lethal method for hemolymph collection and dilution; 2) verify the capability of flow cytometry for hemocyte count and type identification through comparison with microscopic observation; 3) validate hemocyte viability assay based on plasma membrane integrity, and 4) develop hemocyte phagocytosis assay by use of fluorescein labeled microbeads. A non-lethal hemocyte collection method was developed using needle insertion through the ligament. Osmolality measurement of serum was the same as that of culture seawater. The pH measurement of serum (7.2) was significantly different from that of culture seawater (8.4). By microscopic observation, three types of hemocytes were identified with granulocytes, the dominant cell type (70 ± 16%), agranulocyte (14 ± 4%), and blast-like cell (16 ± 4%), and no differences were found from the measurements by flow cytometer on FSC/SSC plot (cell size/granularity). The viability of hemocytes based on plasma membrane integrity was 88 ± 6% ranging from 70 to 97% (n = 60, three populations), and viability protocol was further validated with the pre-set expected viability (p ≥ 0.424). Phagocytosis assay of hemocytes with fluorescence beads showed a mean capacity of 10 ± 5% (n = 60, three populations). Incubation time (up to 6 h) or bead concentrations (2:1 or 5:1 to hemocytes) did not affect the phagocytosis measurement. Overall, this study reported the basic characteristics of hemolymph (serum and hemocytes) of northern quahogs. It is expected that the assay methodologies will be applied to evaluation of hemocyte responses to environmental stresses for clam aquaculture.

Identification of clam plasma proteins that bind its pathogen Quahog Parasite Unknown.

The hard clam (Mercenaria mercenaria) is among the most economically-important marine species along the east coast of the United States, representing the first marine resource in several Northeastern states. The species is rather resilient to infections and the only important disease of hard clams results from an infection caused by Quahog Parasite Unknown (QPX), a protistan parasite that can lead to significant mortality events in wild and aquacultured clam stocks. Though the presence of QPX disease has been documented since the 1960s, little information is available on cellular and molecular interactions between the parasite and the host. This study examined the interactions between the clam immune system and QPX cells. First, the effect of clam plasma on the binding of hemocytes to parasite cells was evaluated. Second, clam plasma proteins that bind QPX cells were identified through proteomic (LC-MS/MS) analyses. Finally, the effect of prior clam exposure to QPX on the abundance of QPX-reactive proteins in the plasma was evaluated. Results showed that plasma factors enhance the attachment of hemocytes to QPX. Among the proteins that specifically bind to QPX cells, several lectins were identified, as well as complement component proteins and proteolytic enzymes. Furthermore, results showed that some of these lectins and complement-related proteins are inducible as their abundance significantly increased following QPX challenge. These results shed light on plasma proteins involved in the recognition and binding of parasite cells and provide molecular targets for future investigations of factors involved in clam resistance to the disease, and ultimately for the selection of resistant clam stocks.

Clam focal and systemic immune responses to QPX infection revealed by RNA-seq technology.

BACKGROUND: The hard clam Mercenaria mercenaria is an important seafood species widely exploited along the eastern coasts of the United States and play a crucial role in coastal ecology and economy. Severe hard clam mortalities have been associated with the protistan parasite QPX (Quahog Parasite Unknown). QPX infection establishes in pallial organs with the lesions typically characterized as nodules, which represent inflammatory masses formed by hemocyte infiltration and encapsulation of parasites. QPX infection is known to induce host changes on both the whole-organism level and at specific lesion areas, which imply systemic and focal defense responses, respectively. However, little is known about the molecular mechanisms underlying these alterations. RESULTS: RNA-seq was performed using Illumina Hiseq 2000 (641 Million 100 bp reads) to characterize M. mercenaria focal and systemic immune responses to QPX. Transcripts were assembled and the expression levels were compared between nodule and healthy tissues from infected clams, and between these and tissues from healthy clams. De novo assembly reconstructed a consensus transcriptome of 62,980 sequences that was functionally-annotated. A total of 3,131 transcripts were identified as differentially expressed in different tissues. Results allowed the identification of host immune factors implicated in the systemic and focal responses against QPX and unraveled the pathways involved in parasite neutralization. Among transcripts significantly modulated upon host-pathogen interactions, those involved in non-self recognition, signal transduction and defense response were over-represented. Alterations in pathways regulating hemocyte focal adhesion, migration and apoptosis were also demonstrated. CONCLUSIONS: Our study is the first attempt to thoroughly characterize M. mercenaria transcriptome and identify molecular features associated with QPX infection. It is also one of the first studies contrasting focal and systemic responses to infections in invertebrates using high-throughput sequencing. Results identified the molecular signatures of clam systemic and focal defense responses, to collectively mediate immune processes such as hemocyte recruitment and local inflammation. These investigations improve our understanding of bivalve immunity and provide molecular targets for probing the biological bases of clam resistance towards QPX.

Interactive effects of copper exposure and environmental hypercapnia on immune functions of marine bivalves Crassostrea virginica and Mercenaria mercenaria.

Estuarine organisms such as bivalves are commonly exposed to trace metals such as copper (Cu) and hypercapnia (elevated CO2 levels) in their habitats, which may affect their physiology and immune function. This study investigated the combined effects of elevated CO2 levels (∼800-2000 µatm PCO2, such as predicted by the near-future scenarios of global climate change) and Cu (50 µg l(-1)) on immune functions of the sediment dwelling hard clams Mercenaria mercenaria and an epifaunal bivalve, the eastern oyster Crassostrea virginica. Clams and oysters were exposed for 4 weeks to different CO2 and Cu levels, and tissue Cu burdens and immune parameters were assessed to test the hypothesis that hypercapnia will enhance Cu uptake due to the higher bioavailability of free Cu(2+) and increase the immunomodulatory effects of Cu. Exposure to Cu stimulated key immune parameters of clams and oysters leading to increased number of circulating hemocytes, higher phagocytosis and adhesion ability of hemocytes, as well as enhanced antiparasitic and antibacterial properties of the hemolymph reflected in higher activities of lysozyme and inhibitors of cysteine proteases. Lysozyme activation by Cu exposure was most prominent in normocapnia (∼400 µatm PCO2) and an increase in the levels of the protease inhibitors was strongest in hypercapnia (∼800-2000 µatm PCO2), but other immunostimulatory effects of Cu were evident in all PCO2 exposures. Metabolic activity of hemocytes of clams and oysters (measured as routine and mitochondrial oxygen consumption rates) was suppressed by Cu exposure likely reflecting lower rates of ATP synthesis and/or turnover. However, this metabolic suppression had no negative effects of the studied immune functions of hemocytes such as phagocytosis or adhesion capacity. Hypercapnia (∼800-2000 µatm PCO2) slightly but significantly enhanced accumulation of Cu in hemocytes, consistent with higher Cu(2+) bioavailability in CO2-acidified water, but had little effect on cellular and humoral immune traits of clams and oysters. These findings indicate that low levels of Cu contamination may enhance immunity of estuarine bivalves while moderate hypercapnia (such as predicted by the near future scenarios of the global climate change) does not strongly affect their immune parameters.

Immunomodulation by the interactive effects of cadmium and hypercapnia in marine bivalves Crassostrea virginica and Mercenaria mercenaria.

Estuarine organisms are exposed to multiple stressors including large fluctuations in partial pressure of carbon dioxide (P2CO) and concentrations of trace metals such as cadmium (Cd) that can affect their survival and fitness. Ocean acidification due to the increasing atmospheric (P2CO) leads to a decrease in pH and shifts in the carbonate chemistry of seawater which can change bioavailability and toxicity of metals. We studied the interactive effects of (P2CO) and Cd exposure on metal levels, metabolism and immune-related functions in hemocytes of two ecologically and economically important bivalve species, Mercenaria mercenaria (hard shell clam) and Crassostrea virginica (Eastern oyster). Clams and oysters were exposed to combinations of three (P2CO) levels (∼400, 800 and 2000 µatm (P2CO), corresponding to the present day conditions and the projections for the years 2100 and 2250, respectively) and two Cd concentrations (0 and 50 µg l(-1)) in seawater. Following four weeks of exposure to Cd, hemolymph of both species contained similar Cd levels (50-70 µg l(-1)), whereas hemocytes accumulated intracellular Cd burdens up to 15-42 mg l(-1), regardless of the exposure P2CO. Clam hemocytes had considerably lower Cd burdens than those of oysters (0.7-1 ng 10(-6) cells vs. 4-6 ng 10(-6) cells, respectively). Cd exposure suppressed hemocyte metabolism and increased the rates of mitochondrial proton leak in normocapnia indicating partial mitochondrial uncoupling. This Cd-induced mitochondrial uncoupling was alleviated in hypercapnia. Cd exposure suppressed immune-related functions in hemocytes of clams and oysters, and these effects were exacerbated at elevated (P2CO). Thus, elevated (P2CO) combined with Cd exposure resulted in decrease in phagocytic activity and adhesion capacity as well as lower expression of mRNA for lectin and heat shock protein (HSP70) in clam and oyster hemocytes. In oysters, combined exposure to elevated (P2CO) and Cd also led to reduced activity of lysozyme in hemocytes and hemolymph. Overall, our study shows that moderately elevated (P2CO) (∼800-2000 µatm P2CO) potentiates the negative effects of Cd on immunity and thus may sensitize clams and oysters to pathogens and diseases during seasonal hypercapnia and/or ocean acidification in polluted estuaries.

Effects of salinity on hard clam (Mercenaria mercenaria) defense parameters and QPX disease dynamics.

QPX (Quahog Parasite Unknown) is a protistan parasite affecting hard clams (Mercenaria mercenaria) along the Northeast coast of the United States. The fact that QPX disease epizootics are usually observed in field sites with high salinities led to the general assumption that salinity represents an important factor for disease distribution. This study was designed to investigate the effect of salinity on QPX disease development as well as constitutive and QPX-induced defense factors in M. mercenaria. Naïve and QPX-infected (both experimentally and naturally) clams were submitted to 17 and 30 psu for 4 months. Standard and QPX-specific cellular and humoral defense parameters were assessed after 2 and 4 months. These included total and differential hemocyte counts, reactive oxygen species production, phagocytic activity of hemocytes, lysozyme concentration in plasma, anti-QPX activity in plasma and resistance of hemocytes to cytotoxic QPX extracellular products. Results demonstrated higher QPX-associated mortality in naturally infected clams maintained at high salinity compared to those held at 17 psu. Our findings also showed an increase in mortality following experimental challenge with QPX in clams submitted to 30 psu but not in those held at 17 psu. Constitutive clam defense factors and the response to QPX challenge were also affected by salinity. QPX challenge caused significant but transitory changes in hemolymph parameters that were obvious at 2 months but disappeared at 4 months. Overall, our results show that salinity modulates clam immunity and the progress of QPX disease although its impact appears secondary as compared to findings we reported earlier for temperature.

Effects of temperature on hard clam (Mercenaria mercenaria) immunity and QPX (Quahog Parasite Unknown) disease development: I. Dynamics of QPX disease.

Quahog Parasite Unknown (QPX) causes disease and mortality in hard clams, Mercenaria mercenaria. Seasonality of QPX disease prevalence in the field and changes in QPX growth and survival in vitro suggest a role of temperature in the hard clam-QPX interaction and disease development. This study specifically examined the effect of temperature on QPX disease development and dynamics. Naturally and experimentally infected clams were separately maintained in the laboratory at 13°C, 21°C, or 27°C for 4 months. Following this initial treatment, temperature was adjusted to 21°C for 5 additional months to simulate seasonal changes of temperature in the field and to investigate the effect of temperature variations on QPX disease dynamics. Mortality was continuously monitored during the experiment and clams were sampled at 2, 4 and 9 months for the assessment of QPX disease prevalence and intensity using our standard histological and quantitative PCR techniques. Results demonstrated significantly higher QPX disease prevalence and intensity, as well as higher mortality, in naturally-infected clams maintained at 13°C as compared to those held at 21°C or 27°C. Similarly, disease development was significantly higher in experimentally infected clams maintained at the colder temperature (70% prevalence after 4 months) as compared to those maintained under warmer conditions (<10%). Additionally, our results demonstrated an improvement in the condition of clams initially maintained at 13°C for 4 months after transfer to 21°C for 5 additional months, with a significant reduction of QPX prevalence (down to 19%). Interestingly, disease development or healing in clams maintained at different temperatures exhibited a strong relationship with clam defense status (jointly submitted paper) and highlighted the impact of temperature on clam activity and QPX disease dynamics. These findings should be taken into account for the timing of activities involving the monitoring, movement (e.g. relays, transplants) or grow out (e.g. commercial culture, municipal enhancement) of hard clams in enzootic areas.

Effects of temperature on hard clam (Mercenaria mercenaria) immunity and QPX (Quahog Parasite Unknown) disease development: II. Defense parameters.

Quahog Parasite Unknown (QPX) is a protistan parasite affecting hard clams Mercenaria mercenaria along the Northeastern coast of the United States. The geographic distribution and occurrence of disease epizootics suggests a primary role of temperature in disease development. This study was designed to investigate the effect of temperature on constitutive and QPX-induced defense factors in M. mercenaria. Control and QPX-challenged (both experimentally and naturally) clams were maintained at 13, 21 and 27°C for 4 months. Control and experimentally-infected clams originated from a southern broodstock (Florida, no prior reports of disease outbreak) while naturally-infected clams originated from a northern broodstock (Massachusetts, enzootic area). Standard and QPX-specific cellular and humoral defense parameters were assessed after 2 and 4 months. Measured parameters included total and differential hemocyte counts, reactive oxygen species production, phagocytic activity of hemocytes, lysozyme concentration in plasma, anti-QPX activity in plasma and resistance of hemocytes to cytotoxic QPX extracellular products. Results demonstrated a strong influence of temperature on constitutive clam defense factors with significant modulation of cellular and humoral parameters of control clams maintained at 13°C compared to 21 and 27°C. Similarly, clam response to QPX challenge was also affected by temperature. Challenged clams exhibited no difference from controls at 27°C whereas different responses were observed at 21°C and 13°C compared to controls. Despite differences in infection mode (experimentally or naturally infected) and clam origin (northern and southern broodstocks), similarities were observed at 13°C and 21°C between QPX infected clams from Florida and Massachusetts. Clam response to temperature and to QPX exhibited interesting relationship with QPX disease development highlighting major influence of temperature on disease development.