In vitro interactions between several species of harmful algae and haemocytes of bivalve molluscs

Harmful algal blooms (HABs) can have both lethal and sublethal impacts on shellfish. To understand the possible roles of haemocytes in bivalve immune responses to HABs and how the algae are affected by these cells (haemocytes), in vitro tests between cultured harmful algal species and haemocytes of the northern quahog (= hard clam) Mercenaria mercenaria, the soft-shell clam Mya arenaria, the eastern and Pacific oysters Crassostrea virginica and Crassostrea gigas and the Manila clam Ruditapes philippinarum were carried out. Within their respective ranges of distribution, these shellfish species can experience blooms of several HAB species, including Prorocentrum minimum, Heterosigma akashiwo, Alexandrium fundyense, Alexandrium minutum and Karenia spp.; thus, these algal species were chosen for testing. Possible differences in haemocyte variables attributable to harmful algae and also effects of haemolymph and haemocytes on the algae themselves were measured. Using microscopic and flow cytometric observations, changes were measured in haemocytes, including cell morphology, mortality, phagocytosis, adhesion and reactive oxygen species (ROS) production, as well as changes in the physiology and the characteristics of the algal cells, including mortality, size, internal complexity and chlorophyll fluorescence. These experiments suggest different effects of the several species of harmful algae upon bivalve haemocytes. Some harmful algae act as immunostimulants, whereas others are immunosuppressive. P. minimum appears to activate haemocytes, but the other harmful algal species tested seem to cause a suppression of immune functions, generally consisting of decreases in phagocytosis, production of ROS and cell adhesion and besides cause an increase in the percentage of dead haemocytes, which could be attributable to the action of chemical toxins. Microalgal cells exposed to shellfish haemolymph generally showed evidence of algal degradation, e.g. loss of chlorophyll fluorescence and modification of cell shape. Thus, in vitro tests allow a better understanding of the role of the haemocytes and the haemolymph in the defence mechanisms protecting molluscan shellfish from harmful algal cells and could also be further developed to estimate the effects of HABs on bivalve molluscs in vivo.     

Flow cytometric analysis of crayfish haemocytes activated by lipopolysaccharides

Lipopolysaccharides (LPS) from Gram-negative bacteria are strong stimulators of white river crayfish, Procambarus zonangulus, haemocytes in vitro. Following haemocyte treatment with LPS and with LPS from rough mutant R5 (LPS Rc) from Salmonella minnesota, flow cytometric analysis revealed a conspicuous and reproducible decrease in cell size as compared to control haemocytes. These LPS molecules also caused a reduction in haemocyte viability as assessed by flow cytometry with the fluorescent dyes calcein-AM and ethidium homodimer. The onset of cell size reduction was gradual and occurred prior to cell death. Haemocytes treated with LPS from S. minnesota without the Lipid A moiety (detoxified LPS) decreased in size without a reduction of viability. The action of LPS on crayfish haemocytes appeared to be related to the activation of the prophenoloxidase system because phenoloxidase (PO)-specific activity in the supernatants from control and detoxified LPS-treated cells was significantly lower than that from LPS and LPS-Rc treated cells (P相似文献   

Phenoloxidase activity in larval and juvenile homogenates and adult plasma and haemocytes of bivalve molluscs

Phenoloxidase (PO) activity was studied in larval and juvenile homogenates and in the plasma and haemocytes of adult Crassostrea gigas, Argopecten ventricosus, Nodipecten subnodosus, and Atrina maura. Samples were tested for the presence of PO activity by incubation with the substrate L-3, 4-dihydroxyphenylalanine using trypsin, alpha-chymotrypsin, laminarin, lipopolysaccharides (LPS), and sodium dodecyl sulphate (SDS) to elicit activation of prophenoloxidase (proPO) system. PO activity was not detected in larval homogenate. In juvenile homogenate, PO activity was found only in C. gigas and N. subnodosus. PO activity was present in adult samples and was enhanced by elicitors in the plasma of all species tested, but in haemocyte lysate supernatant (HLS) of only N. subnodosus. Activation of proPO by laminarin was suppressed by a protease inhibitor cocktail (P-2714) in plasma and HLS of all species tested.     

Crystalline style proteins from bivalve molluscs

• 1.1. Proteins from crystalline styles of twelve species of bivalve mollusc were examined under different gel electrophoresis conditions and stained to reveal both protein and carbohydrate.
• 2.2. Native extracts of styles produced relatively few protein bands, however denaturation with SDS resulted in much more complex zymograms.
• 3.3. All species possessed several prominent high mol wt glycoproteins.
• 4.4. Eulamellibranchia all had a major non-glycosylated protein at approx. 62,000 mol. wt.
• 5.5. Most Filibranchia had a major non-glycosylated protein at 37,000–50,000 mol. wt.
• 6.6. Eulamellibranchia were a much more homogeneous group than the Filibranchia.

     

Comparison of antigenicity among haemocytes of seven bivalve species by monoclonal antibodies against haemocytes of scallop (Chlamys farreri)

In this paper, haemocyte antigenicity of seven bivalve species (scallop (Chlamys farreri), bay scallop (Argoecten irradians), oyster (Crassostrea talienwhanensis), asiatic hard clam (Meretrix meretrix), monila clam (Ruditapes philipinarum), purplish washington clam (Saxidomus purpuratus) and horny ark (Scapharca subcrenta)) were analysed using monoclonal antibodies (MAbs) 1E7, 1F12, 2C6 and 2H5 against haemocytes of C. farreri, employed methods of immuno-dotblotting (IDB), indirect immunofluorescence assay (IIFA) and western-blotting (WB). The four MAbs react with haemocytes of seven bivalve species. As the results for both IDB and IIFA, MAb 1E7 was positive with haemocytes of R. philipinarum, MAb 1F12 with haemocytes of A. irradians, M. meretrix, R. philipinarum and S. purpuratus; MAb 2C6 with haemocytes of the other five bivalve species except for S. purpuratus. MAb 2H5 was negative with haemocytes of the other six bivalve species in IDB, but was positive with haemocytes of R. philipinarum and S. purpuratus in IIFA. Further experiments by WB showed MAb 1F12 was able to recognise the protein of A. irradians haemocyte at molecular weights of 156 and 80 kDa, haemocytes of M. meretris, R. philipinarum, S. purpuratus, at 60, 30, 58 kDa, respectively. MAb 2C6 recognised haemocyte M. meretris proteins at 50 and 37 kDa, A. irradians, C. talienwhanensis, R. philipinarum, S. subcrenta at 40, 38, 38, 45 kDa, respectively. There were no protein bands reacting with MAb 1E7 and MAb 2H5. The results indicate antigenic similarities exist among haemocytes of the seven bivalve species.     

Flow cytometric and karyological analyses of Calendula species from Iberian Peninsula

Calendula L. (Asteraceae) is a taxonomically and cytologically complex genus due to its high morphological and karyological variation. To gather consistent cytological information aiming to consolidate the existing knowledge, sustain the taxonomic revision of the genus and explore the evolutionary relationships among species, the genome size and chromosome number of the Iberian Peninsula representatives of this genus were assessed. The study included 11 taxa that occur in the Iberian Peninsula, one in Madeira and two from Morocco. Chromosome counts were made using the squash technique in root tips and flower buds, while nuclear DNA contents were assessed using propidium iodide flow cytometry. The following chromosome numbers are reported: 2n = 44 for C. arvensis, 2n = 30 for C. tripterocarpa, and 2n = 32 for the remaining Iberian taxa. The genome size of Calendula species was assessed for the first time and ranged from 1.75 pg/2C in C. maroccana to 5.41 pg/2C in C. arvensis. Within the complex formed by C. incana and C. suffruticosa, a gradient of genome size values was obtained. Intraspecific variation in genome size was detected in some taxa. The obtained genome size values and their variation are discussed in the light of the theories proposed for the speciation of the genus, with events of hybridization, genome duplication and dysploidy being hypothesized to play a major role in the evolution of this genus.     

Biotransformations of paralytic shellfish toxins by bacteria isolated from bivalve molluscs

Due to the possibility that bacteria could be involved in the clearance of paralytic shellfish toxins (PST) from bivalve molluscs, investigations into which, if any, bacteria were able to grow at the expense of PST focused on several common shellfish species. These species were blue mussels, oysters, razor fish, cockles, and queen and king scallops. Bacteria associated with these shellfish were isolated on marine agar 2216 and characterized by their carbon utilization profiles (BIOLOG). Selected isolates from groups demonstrating 90% similarity were screened for their ability to metabolize a range of PST (gonyautoxins 1 and 4 [GTX 1/4], GTX 2/3, GTX 5, saxitoxin, and neosaxitoxin) using a novel screening method and confirming its results by high-performance liquid chromatography. Results suggest that molluscan bacteria have different capacities to utilize and transform PST analogues. For example, isolates M12 and R65 were able to reductively transform GTX 1/4 with concomitant production of GTX 2/3, while isolate Q5 apparently degraded GTX 1/4 without the appearance of other GTXs. Other observed possible mechanisms of PST transformations include decarbamoylation by isolate M12 and sulfation of GTXs by isolates Q5, R65, M12, and C3. These findings raise questions as to the possible role of bacteria resident in the shellfish food transport system. Some researchers have suggested that the microflora play a role in supplying nutritional requirements of the host. This study demonstrates that bacteria may also be involved in PST transformation and elimination in molluscan species.     

The capacity for intracellular osmoregulation mediated by free amino acids in three bivalve molluscs

The capacity for intracellular osmoregulation mediated by free amino acids was examined using isolated foot preparations of the three bivalve molluscs: the oligohalineCorbiculajaponica Prime, freshwater euryhalineC. leana Prime, and freshwater stenohalineAnodonta woodiana Lea species. In response to a salinity increase in the incubation medium, ninhydrin-positive substances accumulated in the isolated foot of the oligohaline and freshwater euryhaline species, but not in the freshwater stenohaline species. Possible explanations for such a difference were considered along with its evolutionary significance regarding the entrance of bivalves into the freshwater environment.     

Ciliary activity on the ctenidium of bivalve molluscs

1. Lateral ciliary activity was studied on the ctenidial preparations of several bivalves.2. The cerebral and visceral ganglia exhibit a coordinated role in the control of ciliary beating.3. Exposure of the ctenidia to changes in potassium and magnesium ion concentrations at acclimation salinities were salinity-dependent and probably reflect an effect on the ciliated epithelium.4. The magnitude of cilio-inhibition is directly related to the percentage of lamellar conversions in cytosomes.5. Since sequestered calcium has been shown to be released from lamellar-type cytosomes, it is postulated that lateral cilio-inhibition is due to an increase in the neuronal intracellular calcium concentration with subsequent release of the cilio-inhibitory neurotransmitter, dopamine.6. There is a seasonal effect on cytosomal transformations and decarboxylase activity in neuronal tissues both in the central and peripheral elements and lateral ciliary activity.7. The experimental design and procedures used in our studies will have broad applications for quantitatively assessing the effects of environmental factors on ciliary activity of marine and estuarine organisms.     

Isolation and structural studies of heparan sulfates and chondroitin sulfates from three species of molluscs

The isolation and some structural features of heparan sulfates and chondroitin sulfates from three species of molluscs (Pomacea sp., Tagelus gibbus, and Anomalocardia brasiliana) are reported. It is shown that heparan sulfates with structural similarities to the ones found in mammals are present in the three molluscs analyzed. All the heparan sulfates were degraded by heparitinases I and II to four distinct unsaturated disaccharides with the same properties as the ones present in heparan sulfates of mammalian origin. This suggests that these four disaccharide units are maintained through the evolution. Furthermore, the proportion of these units varied in the heparan sulfates according to the species of origin. The chondroitin sulfates, on the other hand, exhibit different structural features according to the species of origin. For instance, by the action of chondroitinase AC, 4- and nonsulfated disaccharides are produced from Pomacea chondroitin, whereas 4- and 6-sulfated disaccharides are formed from Tagelus and Anomalocardia. The possible role of these compounds in cell recognition and/or adhesiveness is discussed in view of the present findings.     

The adenosine triphosphate content of some marine bivalve molluscs

The total ATP-content of representatives of 23 bivalve families was found to vary from 1.26–0.26 % of the dry tissue weight, with the dry tissue containing about 40 % carbon. Representatives from certain families consistently had more than average and others less than average ATP values, and there were greater differences between species from different families than between closely related species. Variations in the oxygen uptake of whole animals accounted for little of the observed variation in ATP-content. The ATP-content of individual tissues from the same individual showed wide variation, as did that of the same tissue from different species. The highest values of ATP were found in muscle tissues. High maintained levels of ATP were associated with the ability of the species to use energy rapidly for short periods, for example, in escape responses.     

Anaerobic metabolism of bivalve molluscs during exposure to air

The anaerobic metabolism of Mytilus edulis, Cardium edule, Scrobicularia plana and Macoma balthica was investigated. On exposure to the atmosphere, all of these species were found to be able to utilise ¹⁴CO₂. This suggests that these species gape during exposure to the atmosphere. A comparative study on the pattern of ¹⁴C incorporation suggests that there is a similarity between M. edulis and S. plana in the extent of the utilization of the succinate pathway during exposure to air. However C. edule and M. balthica were also similar in the extent of utilizing the succinate pathway, even though there were significant species differences between the similar animals. It is suggested that exposure in S. plana represents a stressful situation and that this species might react to this stress by utilizing the succinate pathway. A higher incorporation of radiocarbon into alanine by M. balthica could be due to high activity of the enzymes that control the reactions leading to production of radioactive pyruvate.