Clam Hemolymph Research Articles

Morphological Analysis of the Hemolymph Cell Composition in the Bivalve Mollusk <i>Anadara</i> <i>broughtonii</i> Schrenck, 1867 (Sea of Japan)

The hemolymph cells of the ark clam Anadara broughtonii were examined using light microscopy, flow cytometry and gradient centrifugation. All three methods of analysis made it possible to identify two main types of cells in the hemolymph of the ark clam Anadara broughtonii – large granular erythrocytes and small agra-nular amebocytes. Erythrocytes accounted for 95.6 ± 0.9% of the total number of hemolymph cells. Erythrocytes were hemoglobin-containing cells with a great number of granules in the cytoplasm, a low nuclear-cytoplasmic ratio (NCR) and a lower intensity of cellular respiration compared to amebocytes. Amebocytes are cells of predominantly irregular shape with a high number of cells that do not contain or contain no more than 10 granular inclusions in the cytoplasm. All types of hemocytes found in the ark clam hemolymph demonstrated the same ability to spontaneously produce reactive oxygen species. For the first time, it has been shown that red blood cells of the ark clam Anadara broughtonii are capable of phagocytosis. At the same time, amebocytes absorbed on the average two times more zymosan particles (10.3 ± 0.7 pcs.), compared to erythrocytes (5.3 ± 0.1 pcs.).

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.

Влияние суточной гипоксии на функциональные показатели гемоцитов Anadara kagoshimensis (Tokunaga, 1906)

Марикультурные хозяйства традиционно расположены в прибрежных участках, где моллюски могут подвергаться воздействию гипоксии. Культивирование в условиях дефицита кислорода приводит к снижению темпов роста, вспышкам заболеваний на фермах и массовой гибели моллюсков. Методом проточной цитометрии исследовано влияние краткосрочной гипоксии на функциональные показатели гемоцитов анадары (Anadara kagoshimensis). Контрольную группу содержали при 6,7–6,8 мг O2·л−1, опытную — при 0,4–0,5 мг O2·л−1. Экспозиция — 24 часа. Содержание кислорода в воде снижали, продувая её газообразным азотом. В гемолимфе моллюска на основании относительного размера и относительной гранулярности идентифицировано две группы гемоцитов: гранулоциты (эритроциты) и агранулоциты (амёбоциты). Эритроциты — преобладающий тип клеток в гемолимфе A. kagoshimensis: их доля составила более 90 % от общего числа клеток. Суточная гипоксия не привела к изменениям клеточного состава гемолимфы анадары. Способность к продукции активных форм кислорода и уровень смертности гемоцитов моллюсков экспериментальной группы также остались на уровне контрольных значений. Результаты проведённого исследования свидетельствуют о толерантности анадары к условиям острого дефицита кислорода.

External pH modulation during the growth of Vibrio tapetis, the aetiological agent of brown ring disease.

Brown ring disease (BRD) is an infection of the Manila clam Ruditapes philippinarum due to the pathogen Vibrio tapetis. During BRD, clams are facing immunodepression and shell biomineralization alteration. In this paper, we studied the role of pH on the growth of the pathogen and formulated hypothesis on the establishment of BRD by V. tapetis. In this study, we monitored the evolution of pH during the growth of V. tapetis in a range of pH and temperatures. We also measured the pH of Manila clam haemolymph and extrapallial fluids (EPFs) during infection by V. tapetis. We highlighted that V. tapetis modulates the external pH during its growth, to a value of 7·70. During the development of BRD, V. tapetis also influences EPFs and haemolymph pH in vitro in the first hours of exposure and in vivo after 3days of infection. Our experiments have shown a close interaction between V. tapetis CECT4600, a pathogen of Manila clam that induces BRD, and the pH of different compartments of the animals during infection. These results indicate that the bacterium, through a direct mechanism or as a consequence of physiological changes encountered in the animal during infection, is able to interfere with the pH of Manila clam fluids. This pH modification might promote the infection process or at least create an imbalance within the animal that would favour its persistence. This last hypothesis should be tested in future experiment. This study is the first observation of pH modifications in the context of BRD and might orient future research on the fine mechanisms of pH modulation associated with BRD.

Morphologic, cytometric and functional characterisation of Anadara kagoshimensis hemocytes

Circulating hemocytes of ark clam, Anadara kagoshimensis, were investigated using light microscopy and flow cytometry. Hemolymph contained 3 morphotype of cells, amebocytes, erythrocytes and intermediate type cells, which formed two distinct subpopulations on flow cytometric dot plots. Large cells (intermediate morphotype and erythrocytes) amounted 85.6 ± 2.8% total cells in hemolymph. Erythrocytes were hemoglobin-containing cells with up to 40 granules presented in the cytoplasm. All hemocyte types, observed in the ark clam hemolymph demonstrated equal capacity to spontaneous production of reactive oxygen species.

A metabolomics approach to assess the effect of storage conditions on metabolic processes of New Zealand surf clam (Crassula aequilatera)

Temperature fluctuations during the live storage and transport of bivalves are known to be one of the most important stressors for these markets. However, the biological mechanisms that induce these stresses and the immunological responses to stressors are not currently understood. In this study, a gas chromatography–mass spectrometry (GC–MS based) metabolomics approach was used along with flow cytometry to characterize the responses of clams during aerial exposure in two different storage conditions (ice and no ice) and storage periods (1 h and 6 h). The results showed a significant increase in haemocyte mortality and ROS production in clams exposed to room temperature (no ice) and after 6 h compared to low temperature (with ice) and 1 h, respectively. The metabolite profiles of clam haemolymph in all groups revealed changes of many metabolites between the different temperatures and sampling times. Among them, lactic acid, succinic acid, malic acid, fumaric acid and glutamic acid were identified as significantly affected by both storage condition and period. These results indicate that clam storage stresses produced biological disturbances related to energy metabolism, which was switched from aerobic to anaerobic metabolism. These findings provide insights regarding the specific metabolic consequences of bivalve live storage and transport, which may lead to improved product handling along the supply chain. In addition, this study demonstrates the effective use of metabolomics and flow cytometric approaches on bivalve haemolymph as a non-destructive shelf life assessment method.

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.

Reverse transcriptase activity associated with haemic neoplasia in the soft-shell clam Mya arenaria

Reverse transcriptase (RT) activity has been reported in bivalves affected by haemic neoplasia (HN). Since all retroviruses have RT, detection of RT activity was regarded as evidence for the retroviral etiology of HN. This study investigates the relationship between RT levels and the progress of HN as indicated by percentages of tetraploid cells in soft-shell clams Mya arenaria. The percentages of tetraploid cells were estimated by flow cytometry, and the RT levels were quantified using TaqMan product-enhanced RT (TM-PERT) assay. Results demonstrated that the amount of RT was positively correlated with the percentage of tetraploid cells circulating in clam haemolymph (R2 = 0.974, p < 0.001). Compared to HN-negative clams (<5% tetraploid cells), 2 stages with significantly elevated levels of RT activity were observed: the first stage at approximately 10 to approximately 20% tetraploid cells, and the second at approximately 30 to approximately 80% tetraploid cells (p < 0.01). These data support the well established fact from mammalian models that transformed cells express high levels of non-telomeric RT. The observed increase in RT levels at approximately 30% tetraploidy coincides with previously reported p53 gene expression. Taken together, this could indicate that using RT levels as an indicator of HN, > or = 30% tetraploidy is the stage at which the disease process undergoes a change, and perhaps becomes irreversible.

Alteration of acetylcholinesterase activity in Semele solida (MOLLUSCA: SEMELIDAE) as a biochemical response to coastal anthropogenic impact

Acetylcholinesterase (AChE) activity was analyzed as a molecular marker indicative of exposure to organophosphorus pesticide residues in individuals of the endemic clam species Semele solida in selected coastal locations of Chile's VIII Region. AChE activity was assayed in clams from (i) Penco Beach near the mouth of the Andalién River (Concepción Bay), (ii) Lenga Beach, near the mouth of the Lenga Estuary (San Vicente Bay), and (iii) Coliumo Beach, near the mouth of the Coliumo Estuary (Coliumo Bay). We also analyzed variations in protein content of clam hemolymph, and variability in the activity of AChE in relation to the sizes of the individual clams sampled. Collection of the clams was done using routine methods, during the spring of 2005, the period during which the use of pesticides is typically intensified in the surrounding forestry and agriculture. The results showed no significant correlation of AChE activity with either the size of the clam, or with the concentration of proteins in the hemolymph. The lowest AChE activity was observed in clams from mouth of the Andalién River (187.5 ± 34.9 Umin−1) which was significantly less than that measured in specimens collected near the mouth of the Coliumo Estuary and the mouth of the Lenga Estuary. A very close relation was observed between the degree of anthropogenic inputs, ocean dynamics, and alterations in AChE activity in S. solida. This clam appears to be a useful indicator species, and AChE activity a sensitive marker for the presence of xenobiotics.

Use of molecular markers for species identification of Korean Perkinsus sp. isolated from Manila clams Ruditapes philippinarum

Perkinsus is the pathogen responsible for mass mortality of the Manila clam Ruditapes philippinarum. Perkinsus sp. isolated from Manila clams collected in Korean waters was assayed by polymerase chain reaction (PCR) to determine its phylogenetic affinity with other congeneric species. Regions of rRNA of Perkinsus sp. isolated from clam haemolymph were cloned and sequenced. Sequences of a non-transcribed spacer (NTS), internal transcribed spacers (ITS 1, 2) and 5.8S rRNA genes were compared to those available from other Perkinsus species. The NTS sequence of Korean Perkinsus was approximately 99.9 to 100% similar to that of P. atlanticus and 98.06 to 98.15% and 73.05 to 73.14% similar to those of P. olseni and P. marinus, respectively. The ITS 1, 5.8S rRNA and ITS 2 sequences of Korean Perkinsus showed 100% similarity to P. atlanticus and Perkinsus sp. reported from Japan. The ITS-5.8S rRNA sequences of Korean Perkinsus were 99.86 and 93.73% similar to those of P. olseni and P. marinus, respectively. The sporulation pattern and morphology of the Korean Perkinsus were very similar to those of P. atlanticus. Our data suggest that the Perkinsus sp. isolated from clams in Korean waters is P. atlanticus, which is currently synonymous with P. olseni reported from Australia. By considering that P. olseni has taxonomic priority, Korean Perkinsus sp. is accepted as P. olseni (atlanticus).

Determination of vitellogenin-like properties inMya arenaria hemolymph (Saguenay Fjord, Canada): A potential biomarker for endocrine disruption

The level of vitellogenin-like (Vg-like) proteins in the hemolymph of the soft-shell clam (Mya arenaria) was indirectly determined using an alkali-labile phosphate (ALP) measurement assay. Biochemical characterization of hemolymph from actively reproducing clams showed the presence of Zn, Ca and of substantial amounts of ALP, all of which are indicative of fish serum vitellogenin, reported to be a Zn and Ca containing glycolipophosphoprotein. ALP assays undertaken on different phosphate-containing compounds (phosphate-buffer saline, diphospholipids and 12,000×g supernatants of Vg-rich trout ovary homogenates) showed little interference by the first two substrates and confirmed the specificity of the ALP assay as an indirect method of determining Vg-like proteins in clam hemolymph. Moreover, clams injected with 17β-estradiol and two known xenoestrogens (nonylphenol and pentachlorophenol) significantly induced hemolymph ALP levels. Field work revealed significant differences in hemolymph ALP levels from clams collected at specific intertidal stations of the Saguenay Fjord during temporal (May to October) and spatial (June) surveys in 1997, which appear to indicate that (anti-)estrogenic chemicals may be present in the Saguenay Fjord. Our preliminary results thus show that this simple and rapid ALP assay holds promise as a biomarker of estrogen disruption with molluscan shellfish as bioindicators. ©1999 John Wiley & Sons, Inc. Environ Toxicol 14: 455–465, 1999

Parasitism by a Protozoan in the Hemolymph of the Giant Clam,Tridacna crocea

A parasitism by a protozoan was found in the giant clam,Tridacna crocea.The parasites were spindle-shaped, 8.6 ± 0.5 μm in length and 2.5 ± 0.3 μm in width. Structural features of the apical complex of the parasite and a molecular phylogenetic analysis of its 18S rRNA gene sequence indicate that the protozoan belongs to the Apicomplexa. No flagellum was observed in the parasitic protozoan. It infected the eosinophilic granular hemocyte, one of the three types of hemocytes in the clam hemolymph, but it is not known whether it influenced the growth of the clam.

Characterization and Infectivity of Four Bacterial Strains Isolated from Brown Ring Disease-Affected Clams

Four bacterial strains isolated from carpet-shell clams (Ruditapes decussatus) and manila clams (R. philippinarum) exhibiting Brown Ring Disease (BRD) symptoms in Galicia (Northwest of Spain) were characterized using biochemical and serological techniques. Moreover their pathogenicity for clams and resistance against clam hemolymph were determined. All of theVibrioisolates exhibited biochemical characteristics typical of the genus. Strains GR1703RP and GR6301RP, isolated fromR. philippinarumshowing signs of BRD, shared phenotypical and antigenic characteristics and bacterial extracellular products enzymatic activities with theVibrio tapetis.Strains GR0705RD and GR0202RD, isolated fromR. decussatusshowing BRD signs, although biochemically similar toV. tapetis,did not show positive agglutination withV. tapetisantiserum. The virulence of the bacteria varied depending on the bacterial strain and clam species assayed. Mortalities were recorded for a 30-day period, reaching values ranging from 6 to 80%. Strain GR0202RD was the most virulent forR. philippinarumwith a cumulative mortality rate of 45%, while the strain GR0705RD induced the highest prevalence of BRD signs, affecting 75% of the survivors. In the case ofR. decussatus,the four bacterial strains produced a cumulative mortality rate higher than 45%. In general, bacterial strains, showed resistance to clams hemolymph.

Role of carbonic anhydrase in the supply of inorganic carbon to the giant clam?zooxanthellate symbiosis

The mechanism whereby inorganic carbon (Ci) is acquired by the symbiotic association between the giant clam (Tridacna derasa) and zooxanthellae (Symbiodinium sp.) has been investigated. Ci in the haemolymph of the clam is in equilibrium with the surrounding sea water. The photosynthesis rate exhibited by the intact clam varies as a function of the Ci concentration in the clam haemolymph. The gill tissue contains high carbonic anhydrase activity which may be important in adjusting the Ci equilibrium between haemolymph and sea water. Zooxanthellae (Symbiodinium sp.) isolated from the clam mantle prefer CO2 to HCO 3 - as a source of inorganic carbon. The zooxanthellae have low levels of carbonic anhydrase on the external surface of the cell; however, mantle extracts display high carbonic anhydrase activity. Carbonic anhydrase is absent from the mantle of aposymbiotic clams (T. gigas), indicating that this enzyme may be essential to the symbiosis. The enzyme is probably associated with the zooxanthellae tubes in the mantle. The results indicate that carbonic anhydrase plays an important role in the supply of carbon dioxide within the clam symbiosis.

Phagocytosis by hemocytes of the hard clam, Mercenaria mercenaria

Large granular hemocytes of Mercenaria mercenaria avidly phagocytose a variety of biological particles (red blood cells of six species, yeast, and gram-positive and gramnegative bacteria) as well as polystyrene spheres. Clam hemolymph is not necessary for phagocytosis but may have some opsonic effect in certain circumstances (e.g., low temperature and low particle density). Formaldehyde treatment of red blood cells enhances susceptibility to phagocytosis. Phagocytosis by Mercenaria hemocytes in vitro appears to be a nonspecific process.

Respiration of sarcoma cells from the soft-shell clam Mya arenaria L. under various conditions

Oxygen consumption of sarcoma cells from the soft-shell clam Mya arenaria L. was studied. Hemolymph of clams in the terminal stage of sarcoma was strongly hypoxic. In early cases of sarcoma, partial pressure of oxygen in the hemolymph did not differ from the control animals but declined as the sarcoma advanced, and finally fell below the detection limit causing death of sarcomatous clams. Mitotic index declined as the sarcoma progressed and was 100 × lower in hypoxic plasma in the terminal stage than in early cases. Oxygen consumption of the sarcoma cells in 15% salinity seawater (18 °C) was 3.9 × 10 −4 μl· h −1·cell −. Respiration rates declined in salinities < 7.5‰ and > 15% . Temperature stress (37 °C) depressed oxygen consumption before mortality could be detected but returning the cells to 18 °C increased oxygen consumption above the initial level.