Development and validation of a 66K SNP array for the hard clam (Mercenaria mercenaria).

BACKGROUND: The hard clam (Mercenaria mercenaria), a marine bivalve distributed along the U.S. eastern seaboard, supports a significant shellfish industry. Overharvest in the 1970s and 1980s led to a reduction in landings. While the transition of industry from wild harvest to aquaculture since that time has enhanced production, it has also exacerbated challenges such as disease outbreaks. In this study, we developed and validated a 66K SNP array designed to advance genetic studies and improve breeding programs in the hard clam, focusing particularly on the development of markers that could be useful in understanding disease resistance and environmental adaptability. RESULTS: Whole-genome resequencing of 84 individual clam samples and 277 pooled clam libraries yielded over 305 million SNPs, which were filtered down to a set of 370,456 SNPs that were used as input for the design of a 66K SNP array. This medium-density array features 66,543 probes targeting coding and non-coding regions, including 70 mitochondrial SNPs, to capture the extensive genetic diversity within the species. The SNPs were distributed evenly throughout the clam genome, with an average interval of 25,641 bp between SNPs. The array incorporates markers for detecting the clam pathogen Mucochytrium quahogii (formerly QPX), enhancing its utility in disease management. Performance evaluation on 1,904 samples demonstrated a 72.7% pass rate with stringent quality control. Concordance testing affirmed the array's repeatability, with an average agreement of allele calls of 99.64% across multiple tissue types, highlighting its reliability. The tissue-specific analysis demonstrated that some tissue types yield better genotyping results than others. Importantly, the array, including its embedded mitochondrial markers, effectively elucidated complex genetic relationships across different clam groups, both wild populations and aquacultured stocks, showcasing its utility for detailed population genetics studies. CONCLUSIONS: The 66K SNP array is a powerful and robust genotyping tool that offers unprecedented insights into the species' genomic architecture and population dynamics and that can greatly facilitate hard clam selective breeding. It represents an important resource that has the potential to transform clam aquaculture, thereby promoting industry sustainability and ecological and economic resilience.

Nutritional and physicochemical characteristics of Asiatic hard clam powder prepared by different cook-drying processes: a comparative study.

BACKGROUND: Asiatic hard clam (Meretrix meretrix) is an underutilized bivalve resource. This study discusses dried clam powders prepared from this resource to enhance its utilization and improve nutritional security in protein-deficient populations. Dried clam powder was prepared from Asiatic hard clam and the effects of different pre-cooking methods (boil-dried clam powder, BDCP; steam-dried clam powder, SDCP; and microwave-dried clam powder, MDCP) on nutritional (proximate composition, amino acid profiling, mineral profiling, fatty acid profiling) and physicochemical qualities were investigated. RESULTS: Different pre-cooking methods significantly influenced the characteristics of the clam powder. The MDCP sample showed the highest concentration of amino acids, polyunsaturated fatty acids, Na, K, Ca and Mg content compared to BDCP and SDCP. The boiling process led to a loss of nutritional quality in terms of amino acids and macrominerals. The MDCP displayed the highest solubility in water (30.10%) but its oil and water absorption characteristics were the lowest among all the samples. Boil-cooked clam powder displayed the highest oil binding (2.03 mL g-1 protein) capacity. Boiling and steaming processes resulted in malondialdehyde generation compared to microwaving. Different pre-cooking processes did not influence the colour attributes significantly, but the control sample prepared without pre-cooking (CCP) had a significantly lower L* value (32.34), resulting in a darker product. In vitro digestibility of the clam powder varied in the order MDCP > SDCP > BDCP > CCP. CONCLUSION: The study demonstrated that nutritionally rich protein powder can be prepared from Asiatic hard clam. Based on the results, microwave pre-cooking is considered the best pre-cooking method to preserve the nutritional qualities of clam powder. © 2024 Society of Chemical Industry.

Disseminated neoplasia in cultured hard clams (Mercenaria mercenaria).

Marine bivalves are commonly affected by disseminated neoplasia of presumed hemocytic origin (i.e., hemic neoplasia and hemocytic neoplasia). Histopathology of 520 cultured hard clams (Mercenaria mercenaria) from Florida was performed for health surveillance over a consecutive 13-month period. Disseminated neoplasia was identified in 9 of 520 animals (1.7%). The neoplasia was characterized by the presence of large, round to oval, anaplastic cells within hemolymphatic vessels and sinusoids with variable infiltration into adjacent connective tissues of the visceral mass, mantle, foot, and/or adductor muscles. Frequent involvement and/or infiltration of the gill was also identified (5/9). Disseminated neoplasia in other species of clams, mussels, and cockles is considered a transmissible disease. At this time, it is unknown if these hard clams represent de novo development of the disease or potential transmission; however, this report expands both the geographic and host range for this condition.

Comparative analysis of the Mercenaria mercenaria genome provides insights into the diversity of transposable elements and immune molecules in bivalve mollusks.

BACKGROUND: The hard clam Mercenaria mercenaria is a major marine resource along the Atlantic coasts of North America and has been introduced to other continents for resource restoration or aquaculture activities. Significant mortality events have been reported in the species throughout its native range as a result of diseases (microbial infections, leukemia) and acute environmental stress. In this context, the characterization of the hard clam genome can provide highly needed resources to enable basic (e.g., oncogenesis and cancer transmission, adaptation biology) and applied (clam stock enhancement, genomic selection) sciences. RESULTS: Using a combination of long and short-read sequencing technologies, a 1.86 Gb chromosome-level assembly of the clam genome was generated. The assembly was scaffolded into 19 chromosomes, with an N50 of 83 Mb. Genome annotation yielded 34,728 predicted protein-coding genes, markedly more than the few other members of the Venerida sequenced so far, with coding regions representing only 2% of the assembly. Indeed, more than half of the genome is composed of repeated elements, including transposable elements. Major chromosome rearrangements were detected between this assembly and another recent assembly derived from a genetically segregated clam stock. Comparative analysis of the clam genome allowed the identification of a marked diversification in immune-related proteins, particularly extensive tandem duplications and expansions in tumor necrosis factors (TNFs) and C1q domain-containing proteins, some of which were previously shown to play a role in clam interactions with infectious microbes. The study also generated a comparative repertoire highlighting the diversity and, in some instances, the specificity of LTR-retrotransposons elements, particularly Steamer elements in bivalves. CONCLUSIONS: The diversity of immune molecules in M. mercenaria may allow this species to cope with varying and complex microbial and environmental landscapes. The repertoire of transposable elements identified in this study, particularly Steamer elements, should be a prime target for the investigation of cancer cell development and transmission among bivalve mollusks.

Identification of variants associated with hard clam, Mercenaria mercenaria, resistance to Quahog Parasite Unknown disease.

Severe losses in aquacultured and wild hard clam (Mercenaria mercenaria) stocks have been previously reported in the northeastern United States due to a protistan parasite called QPX (Quahog Parasite Unknown). Previous work demonstrated that clam resistance to QPX is under genetic control. This study identifies single nucleotide polymorphism (SNP) associated with clam survivorship from two geographically segregated populations, both deployed in an enzootic site. The analysis contrasted samples collected before and after undergoing QPX-related mortalities and relied on a robust draft clam genome assembly. ~200 genes displayed significant variant enrichment at each sampling point in both populations, including 18 genes shared between both populations. Markers from both populations were identified in genes related to apoptosis pathways, protein-protein interaction, receptors, and signaling. This research begins to identify genetic markers associated with clam resistance to QPX disease, leading the way for the development of resistant clam stocks through marker-assisted selection.

Bioaccumulation of trace elements in the hard clam, Meretrix lyrata, reared downstream of a developing megacity, the Saigon-Dongnai River Estuary, Vietnam.

A large number of white hard clam farms are in the estuary shoreline of Saigon-Dongnai Rivers, which flow through Ho Chi Minh City, a megacity, and numerous industrial zones in the basin catchment area. In this study, eleven trace elements (Mn, Fe, Co, Ni, Cu, Zn, As, Se, Cd, Hg, and Pb) in the hard clam Meretrix lyrata and its habitats including surface water, suspended particulate matter, and sediment were evaluated to understand the bioaccumulation of trace metals from the environment into the whole tissues of the hard clam as well as its different organs. The samples were collected monthly in dry, transition, and wet seasons of the southern part of Vietnam from March to September 2016. The results showed that seasonal and spatial variations of the studied metal concentrations in the hard clam M. lyrata might be influenced by the sea current as well as the surface runoff in the rainy season. The relationship between condition index and the element concentrations in M. lyrata might be affected by the living environment conditions and farming methods. In addition, the hazard index values of all trace elements in the hard clam M. lyrata harvested in the sampling time show that the hard clams farmed in the study area were safe for local consumers.

Modeling the odor-landscape resulting from the pumping behavior of bivalve clams in the presence of predators.

Motivated by experimental findings, a computational fluid dynamics (CFD) model was used to investigate whether the clam Mercenaria mercenaria may alter its cue downstream variability by an exhalant random pumping behavior. This behavior was hypothesized to occur in the presence of predator chemical signals in order to prevent successful tracking by the predator. Simulated downstream flow and mixing conditions derived from the random nature of the clam exhalant jet in a crossflow were analyzed by computing an intermittency factor, determining the field of finite-time Lyapunov exponents (FTLEs) and identifying the resulting Lagrangian coherent structures (LCSs). Numerical simulations illustrate that the effectiveness of a fluctuating exhalant jet to prevent downstream tracking by a crab, depends on the ratio of the exhalant jet to the crossflow. Specifically, the clam could effectively enhance the downstream dispersion to prevent tracking, but only in the range of parameters where LCSs are generated (jet-to-crossflow ratio ≥ 1). Then, the probability of detection is reduced with respect to the case of a less fluctuating exhalant jet.

Alterations of the immune transcriptome in resistant and susceptible hard clams (Mercenaria mercenaria) in response to Quahog Parasite Unknown (QPX) and temperature.

Quahog Parasite Unknown (QPX) is a fatal protistan parasite that causes severe losses in the hard clam (Mercenaria mercenaria) fisheries along the northeastern coast of the US. Field and laboratory studies of QPX disease have demonstrated a major role for water temperature and M. mercenaria genetic origin in disease development. Infections are more likely to occur at cold temperatures, with clam stocks originating from southern states being more susceptible than clams from northern origin where disease is enzootic. Even though the influence of temperature on QPX infection have been examined in susceptible and resistant M. mercenaria at physiological and cellular scales, the underlying molecular mechanisms associated with host-pathogen interactions remain largely unknown. This study was carried out to explore the molecular changes in M. mercenaria in response to temperature and QPX infection on the transcriptomic level, and also to compare molecular responses between susceptible and resistant clam stocks. A M. mercenaria oligoarray (15 K Agilent) platform was produced based on our previously generated transcriptomic data and was used to compare gene expression profiles in naive and QPX-infected susceptible (Florida stock) and resistant (Massachusetts) clams maintained at temperatures favoring disease development (13 °C) or clam healing (21 °C). In addition, transcriptomic changes reflecting focal (the site of infection, mantle) and systemic (circulating hemocytes) responses were also assessed using the oligoarray platform. Results revealed significant regulation of multiple biological pathways by temperature and QPX infection, mainly associated with immune recognition, microbial killing, protein synthesis, oxidative protection and metabolism. Alterations were widely systemic with most changes in gene expression revealed in hemocytes, highlighting the role of circulating hemocytes as the first line of defense against pathogenic stress. A large number of complement-related recognition molecules with fibrinogen or C1q domains were shown to be specially induced following QPX challenge, and the expression of these molecules was significantly higher in resistant clams as compared to susceptible ones. These highly variable immune proteins may be potent candidate molecular markers for future study of M. mercenaria resistance against QPX. Beyond the specific case of clam response to QPX, this study also provides insights into the primitive complement-like system in the hard clam.

Effect of "heat shock" treatments on QPX disease and stress response in the hard clam, Mercenaria mercenaria.

The hard clam, Mercenaria mercenaria, is one of the most valuable commercial mollusk species along the eastern coast of the United States. Throughout the past 2 decades, the hard clam industry in the Northeast was significantly impacted by disease outbreaks caused by a lethal protistan parasite known as Quahog Parasite Unknown (QPX). QPX is an opportunistic pathogen and the infection has been shown to be a cold water disease, where warmer conditions (above 21°C) lead to disease reduction and clam healing. In vitro studies also showed a sharp reduction in parasite growth and survivorship at temperatures exceeding 27°C. In this study, we evaluated the effect of short-term exposures to high temperatures on QPX disease dynamic and clam recovery. Infected clams were collected from an enzootic site and subsequently submitted to one of ten "heat shock" treatments involving a gradient of temperatures and exposure times. QPX prevalence was compared before and 10weeks after heat shock to assess the effect of each treatment on disease progress. Expression of several stress-related genes was measured 1 and 7days after heat shock using qPCR to evaluate the effect of each treatment on clam physiology. Anti-QPX activity in clam plasma was also measured in an attempt to link changes in defense factors to thermal stress and disease progress. Our results suggest that brief exposures to moderate high temperatures promote the greatest remission while imposing the mildest stress to clams. These results are discussed with the aim of providing the industry with possible strategies to mitigate QPX disease.

Bioremediation potential of the hard clam Mercenaria mercenaria as an intensive shrimp aquaculture pond polyculture condidate.

Polyculture practices are important for achieving sustainable aquaculture development. Recently, hard clams polyculture in intensive shrimp ponds has been encouraged because bivalves can consume excess nutrients in aquaculture systems and sequester carbon. To evaluate the bioremediation potential of hard clams polyculture in intensive shrimp ponds, this study built an assessment model based on individual growth models and estimated the potential for nitrogen and phosphorus removal as well as CO2 fixation by hard clams. Firstly, key parameters required for model construction were obtained through field surveys and physiological experiments. Subsequently, an individual growth model for the hard clam Mercenaria mercenaria was developed based on the Dynamic Energy Budget (DEB) theory. Fitting of the growth data indicated that the model accurately replicated the growth patterns of hard clams, with relative root mean square errors of 9.87 % for shell length and 5.02 % for dry tissue weight. Finally, the assessment model for the bioremediation potential of hard clams demonstrated that, over 110 days in the intensive shrimp mariculture pond, the net removal of nitrogen and phosphorus by hard clams were 3.68 kg ha-1 and 0.81 kg ha-1, respectively, and CO2 fixation was 507.00 kg ha-1. These findings suggested that the DEB model is an effective tool for evaluating bivalve ecological remediation potential and can aid in selecting species for sustainable polyculture.

Complete genome sequence of a potential new species Vibrio sp. NTOU-M3 isolated from hard clam, Meretrix taiwanica, in Taiwan.

Vibrio sp. NTOU-M3 is a potential new bacterium isolated from hard clam (Meretrix taiwanica) in the estuarine region of Taiwan. The complete sequences obtained using Oxford Nanopore Technologies and Illumina sequencing consist of a 3,272,438-bp large circular chromosome and a 1,584,497-bp small circular chromosome.

Baseline assessment of microplastics in commercially important marine bivalves from New York, U.S.A.

Microplastic (MP) contamination in bivalve mollusks has become a significant concern over the last few years. These ecologically and economically valuable species are popular seafood items for human consumption. As filter feeders, bivalves may ingest MPs in their bodies, possibly impacting their physiology and fitness. Additionally, a considerable amount of the seafood that humans consume comes from coastal areas where MP concentrations tend to be the highest. This research provides the first examination of MPs in eastern oysters (Crassostrea virginica) and hard clams (Mercenaria mercenaria) that were grown locally in coastal areas of New York, contributing to a baseline for the northeast and mid-Atlantic regions of the U.S. A total of 48 eastern oysters (n = 12 per site, at four sites) and hard clams (n = 24 per site, at two sites) were sampled in summer 2021. While MP fibers and fragments (i.e. polyethylene terephthalate, polystyrene, and polypropylene) were found in some oysters, other contaminants (e.g. indigo dye, phthalocyanine, dye 823, etc.) were found in both bivalve species. Particle composition was verified using Raman microspectroscopy. Although mean MP concentrations were low in eastern oysters (i.e. 0.008 MPs g-1 of soft tissue wet weight; 0.125 MPs ind-1) and not found in hard clams, more research is needed to assess the magnitude of contamination in these edible bivalves.

Fine-scale population structure of the northern hard clam (Mercenaria mercenaria) revealed by genome-wide SNP markers.

Aquaculture is growing rapidly worldwide, and sustainability is dependent on an understanding of current genetic variation and levels of connectivity among populations. Genetic data are essential to mitigate the genetic and ecological impacts of aquaculture on wild populations and guard against unintended human-induced loss of intraspecific diversity in aquacultured lines. Impacts of disregarding genetics can include loss of diversity within and between populations and disruption of local adaptation patterns, which can lead to a decrease in fitness. The northern hard clam, Mercenaria mercenaria (Linnaeus, 1758), is an economically valuable aquaculture species along the North American Atlantic and Gulf coasts. Hard clams have a pelagic larval phase that allows for dispersal, but the level of genetic connectivity among geographic areas is not well understood. To better inform the establishment of site-appropriate aquaculture brood stocks, this study used DArTseq™ genotyping by sequencing to characterize the genetic stock structure of wild clams sampled along the east coast of North America and document genetic diversity within populations. Samples were collected from 15 locations from Prince Edward Island, Canada, to South Carolina, USA. Stringent data filtering resulted in 4960 single nucleotide polymorphisms from 448 individuals. Five genetic breaks separating six genetically distinct populations were identified: Canada, Maine, Massachusetts, Mid-Atlantic, Chesapeake Bay, and the Carolinas (F ST 0.003-0.046; p < 0.0001). This is the first study to assess population genetic structure of this economically important hard clam along a large portion of its native range with high-resolution genomic markers, enabling identification of previously unrecognized population structure. Results of this study not only broaden insight into the factors shaping the current distribution of M. mercenaria but also reveal the genetic population dynamics of a species with a long pelagic larval dispersal period along the North American Atlantic and Gulf coasts.

Oral Zinc-Rich Oyster Supplementation Corrects Anemia in Rats.

This study investigates the impact of various zinc supplementation methods on anemia in rats induced by phenylhydrazine (PHZ) and in 5/6-nephrectomized anemic rats. We compare oral zinc sulfate (ZnSO4) supplementation, oyster Crassostrea gigas supplementation, and hard clam Meretrix lusoria supplementation on red blood cell (RBC) levels. Oral zinc-rich oyster supplementation (2.70 mg Zn (30 g oyster)/day/rat) effectively corrects anemia in both experimental groups. Rats orally fed oysters for four days exhibit similar effectiveness as those receiving a single ZnSO4 injection (0.95 mg Zn (4.18 mg ZnSO4⋅7H2O)/rat). In contrast, oral ZnSO4 supplementation (2.70 mg Zn (11.88 mg ZnSO4⋅7H2O)/day/rat) does not significantly increase RBC levels, suggesting better zinc absorption from oysters. A placebo group of anemic rats supplemented with hard clams, similar in composition to oysters but much lower in zinc, did not change RBC counts. This supports oysters' high zinc content as the key to correcting anemia. Oysters also contain high iron levels, offering a potential solution for iron-deficiency anemia while supporting bone marrow erythropoiesis. In summary, oral oyster supplementation emerges as an effective strategy to correct anemia in rats with added zinc and iron support for erythropoiesis.

Time-course changes in the regulation of ions and amino acids in the hard clam Meretrix lusoria upon lower salinity challenge.

In this study, we examined ion and amino acid regulation in the gill and mantle of the hard clam Meretrix lusoria. We found that the osmolality and Na+ and Cl- concentrations of hard clam hemolymph were significantly reduced after transferring clams from the salinity of their natural habitat [20‰ saltwater (SW)] to a lower salinity environment (10‰ SW). Specific activities of Na+ , K+ -ATPase (NKA), which provides the driving force for the secondary ion transport associated with cell osmoregulation in gills and mantles, were unaffected during the acclimation to lower salinity. In contrast, there was a significant decline in the contents of free amino acids (FAAs) in the gills and mantles of hard clams during lower salinity acclimation. Taurine was established to be the dominant FAA, the content of which is considerably higher than that of other FAAs in the hard clam. Following acclimation to the lower salinity environment, mRNA expression of the taurine transporter (TAUT), which plays a pivotal role in regulating intracellular taurine contents, was significantly upregulated in the gill and downregulated in the mantle of hard clams at different time points. However, the relative abundance of TAUT protein in the gill and mantle was significantly increased after transfer from 20‰ SW to 10‰ SW, which may reflect feedback regulation in response to reduced taurine contents in the gill and mantle of hard clams. Collectively, the findings of this study provide important insights on the dynamic processes of ion and amino acid regulation in the peripheral tissues of bivalves.

Effect of a Novel Microwave-Assisted Induction Heating (MAIH) Technology on the Quality of Prepackaged Asian Hard Clam (Meretrix lusoria).

The microwave-assisted induction heating (MAIH) method-an emerging thermal technique-was studied to heat the prepackaged raw hard clam (Meretrix lusoria). The cooking effects on microbial and physiochemical qualities of clam were investigated. After the heating of the clam meat samples, the aerobic plate count (APC), psychrotrophic bacteria count (PBC), and total volatile basic nitrogen (TVBN) levels decreased with increasing heating time, but the shucking ratio, area shrinkage, and texture (hardness, cohesiveness, and chewiness) increased. In addition, the L* (lightness) and W (whiteness) of the clam meat samples increased significantly at the beginning of the heating period, whereas they decreased significantly with extended heating time. However, a* (redness) had the opposite trend. This study found that when clams were heated for more than 120 s at 130 °C or 150 s at 90 °C, they displayed obvious shrinking and a yellow-brown appearance, indicating that they are overcooked. After heating by MAIH for at least 110 s at 130 °C or 130 s at 90 °C, the samples were cooked well and gains a completely shucking, along with no microbial count detected. Therefore, the results indicated that the optimum heating conditions for prepackaged hard clams subjected to an MAIH machine were 130 °C for 110 s or 90 °C for 130 s.

The complete mitochondrial genome of Meretrix lusoria (Bivalvia: Veneroida: Veneridae) from Kumamoto, Japan.

We sequenced and assembled the complete mitochondrial genome sequence of the Meretrix lusoria, from Kumamoto, Japan. The length of mitogenome is 20,180 bp, including 13 protein-coding genes, two ribosomal RNA genes, and 22 transfer RNA genes. The nucleotide composition of the mitogenome was 25.73% for A, 42.41% for T, 9.35% for C, and 22.49% for G. The AT and GC skewness of mitogenome sequence are -0.245 and 0.412, showing the T-skew and G-skew. The reconstructed phylogenetic relationships of 25 Bivalvia species based on 12 protein-coding genes were highly supported and the clade of all Meretrix clams included had a support value of 99%. Our results shall provide a better understanding in the evolutionary histories of the Veneroida and relative species.

White hard clam (Meretrix lyrata) shells as novel filter media to augment the phosphorus removal from wastewater.

It is well recognized that filter media play a crucial role in constructed wetlands (CWs) for decontamination of phosphorus (P)-rich wastewater. This study investigates the suitability of raw white hard clam shells (WHC) and white hard clam shells thermally modified at 800 °C (WHC-M800) as potential media to enhance P treatment performance in CWs. The results indicated that both WHC and WHC-M800 displayed appropriate physicochemical properties, such as high porosity, excellent hydraulic conductivity, and rich Ca content. WHC-M800 exhibited a superior P adsorption capacity (38.7 mg/g) to WHC (12.8 mg/g). However, the practical utilization of WHC-M800 as filter media in CWs may be compromised, due to certain limitations, for example: extremely high pH values in the post-adsorption solutions; high weight losses during calcination and adsorption processes; low mechanical strength; and intensive energy consumption. In contrast, the WHC demonstrated significant advantages of reasonably high P adsorption capacity, locally abundant availability, low cost, and marginal side effects. The fractionation of inorganic P of WHC and WHC-M800 revealed that Ca-bounded P was the most dominant binding form, followed by loosely bound P, Fe-P, occluded P, and Al-P. The present study demonstrates that recycling of WHC shells as a potential substrate in CWs provides a feasible method for upgrading P removal in CWs. Additionally, it helps to reduce waste WHC shells in a simple, cheap, and eco-friendly way, thus can double environmental benefits.

Hard clam extracts induce atypical apoptosis in human gastric cancer cells.

Hard clams (HCs) are a nutritionally high-quality and popular seafood, and are established to be a potent antitumor food. The aim of the present study was to determine whether HC extracts induce apoptosis in the human gastric cancer cell line, AGS. In contrast with previously reported methods of extraction, crude extracts of HC were obtained by freezing and thawing and by a method free of hot water or organic solvents. The composition, quality and properties of the HC extracts were demonstrated to be stable since the extracts that were evaluated by capillary electrophoresis and HPLC analysis at different timepoints were similar. HC extracts also have an inhibitory effect against the survival of AGS cells. Treatment with HC extracts induced a marked sub-G1 DNA peak and reduced the expression of the anti-apoptotic genes BIRC5 and KPNA2. However, hallmarks of classical apoptosis such as DNA fragmentation and apoptotic body formation were not observed, indicating atypical apoptosis. Furthermore, it was revealed that HC extracts interrupted cell cycle progression in AGS cells through altered expression of six cell cycle-associated genes: CDC20, KPNA2, BIRC5, ANAPC2, CDKN1A and RB1. The present findings suggest that HC may contribute to a novel future anticancer agent.

Ionic and Amino Acid Regulation in Hard Clam (Meretrix lusoria) in Response to Salinity Challenges.

Most marine mollusks are osmoconformers, in that, their body fluid osmolality changes in the direction of the change in environmental salinity. Marine mollusks exhibit a number of osmoregulatory mechanisms to cope with either hypo- or hyperosmotic stress. The effects of changes in salinity on the osmoregulatory mechanisms of the hard clam (Meretrix lusoria, an economically important species of marine bivalve for Taiwan) have not been determined. In this study, we examined the effect of exposure to hypo (10‰)- and hyper (35‰)-osmotic salinity on hard clams raised at their natural salinity (20‰). The osmolality, [Na(+)], and [Cl(-)] of the hard clam hemolymph were changed in the same direction as the surrounding salinity. Further, the contents of total free amino acids including taurine in the gills and mantles were significantly upregulated in hard clam with increasing salinity. The gill Na(+), K(+)-ATPase (NKA) activity, the important enzyme regulating cellular inorganic ions, was not affected by the changed salinity. Mantle NKA activity, however, was stimulated in the 35‰ SW treatment. The taurine transporter (TAUT) is related to the regulation of intracellular contents of taurine, the dominant osmolyte. Herein, a TAUT gene of hard clam was cloned and a TAUT antibody was derived for the immunoblotting. The TAUT mRNA expression of the mantle in hard clam was significantly stimulated in 35‰ SW, but protein expression was not modulated by the changed salinity. In gills of the hard clam with 10‰ SW, both TAUT mRNA and protein expressions were significantly stimulated, and it may reflect a feedback regulation from the decreased gills taurine content under long-term hypoosmotic acclimation. These findings suggest that TAUT expression is regulated differently in gills and mantles following exposure to alterations in environmental salinity. Taken together, this study used the physiological, biochemical and molecular approaches to simultaneously explore the osmoregulation in tissues of hard clam and may further help to understand the osmoregulation in bivalves.