Hard to get, easy to lose: Evolution of mantle photoreceptor organs in bivalves (Bivalvia, Pteriomorphia).

Morphologically diverse eyes have evolved numerous times, yet little is known about how eye gain and loss is related to photic environment. The pteriomorphian bivalves (e.g., oysters, scallops, and ark clams), with a remarkable range of photoreceptor organs and ecologies, are a suitable system to investigate the association between eye evolution and ecological shifts. The present phylogenetic framework was based on amino acid sequences from transcriptome datasets and nucleotide sequences of five additional genes. In total, 197 species comprising 22 families from all five pteriomorphian orders were examined, representing the greatest taxonomic sampling to date. Morphological data were acquired for 162 species and lifestyles were compiled from the literature for all 197 species. Photoreceptor organs occur in 11 families and have arisen exclusively in epifaunal lineages, that is, living above the substrate, at least five times independently. Models for trait evolution consistently recovered higher rates of loss over gain. Transitions to crevice-dwelling habit appear associated with convergent gains of eyespots in epifaunal lineages. Once photoreceptor organs have arisen, multiple losses occurred in lineages that shift to burrowing lifestyles and deep-sea habitats. The observed patterns suggest that eye evolution in pteriomorphians might have evolved in association with light-guided behaviors, such as phototaxis, body posture, and alarm responses.

Hemocyte cell types of the Cortes Geoduck, Panopea globosa (Dall 1898), from the Gulf of California, Mexico.

The geoduck Panopea globosa is an endemic and economic valuable species from the Mexican Northwest coast whose biology has been little studied. No information exists about their hemocytes to date, which is highly important to assess the welfare of wild and cultured organisms. In this study, hemocytes of adult P. globosa were characterized at the morphological, ultrastructural and functional level. The mean number of hemocytes in the hemolymph of P. globosa was 6 × 105 ± 2 × 105 cells mL-1. The cells were identified as granulocytes (Gr) and hyalinocytes (H). The former accounted for 28% of adhered cells in the hemolymph, measured 6-18 µm, showed numerous basophilic granules in the cytoplasm, with round and eccentric nuclei, and a nucleus:cytoplasm ratio of 0.44 ± 0.01. Hyalinocytes were the most abundant cells in the hemolymph of P. globosa (72% adhered cells) and were subdivided, according to their size, in small (Hs) 4-12 µm and large (HL) 6-18 µm. Hyalinocytes were eosinophilic round or ovoid cells with a central or eccentric nucleus, few or no granules in the cytoplasm and similar nucleus:cytoplasm ratio (Hs: 0.63 and HL: 061). Lysosomes and lipids were observed in Gr, while carbohydrates were the most abundant energy substrate in H. Both hemocytic cell types, mainly Gr, were capable to ingest particles and yield superoxide (P > 0.05). The present study shows for the first time the cell types, abundance and immune activities of hemocytes present in the hemolymph of P. globosa. This information provides a useful baseline to carry out further research on the cellular immune response of the clam to potential pathogens or changes in environmental factors.

Mesodesma mactroides Gill Cells Exposed to Copper: Does Hyposmotic Saline Increase Cytotoxicity or Cellular Defenses?

Gill cells of filter feeding mollusks have cellular defense mechanisms, such as multixenobiotic resistance (MXR), that allow them to extrude possible contaminants. To analyze the cytotoxicity and cellular defenses of gills in the clam Mesodesma mactroides, gill cells were exposed to copper in both iso- and hyposmotic solutions. Analysis of MXR activity by fluorescence microscopy showed that hyposmotic saline activated defenses, whereas the presence of copper in isosmotic solution inhibited the activation of defenses. Cell viability was decreased in cells exposed to copper in isosmotic saline, but not in cells exposed to hyposmotic saline. We conclude that when cells cannot defend themselves due to decreased MXR, cell death occurs. In addition, gill cells under hyposmotic conditions have a greater capacity for defense and a lower rate of cellular mortality than when they are maintained under isosmotic conditions.

Effect of acclimatization on hemocyte functional characteristics of the Pacific oyster (Crassostrea gigas) and carpet shell clam (Ruditapes decussatus).

Most experimental procedures on molluscs are done after acclimatization of wild animals to lab conditions. Similarly, short-term acclimation is often unavoidable in a field survey when biological analysis cannot be done within the day of sample collection. However, acclimatization can affect the general physiological condition and particularly the immune cell responses of molluscs. Our aim was to study the changes in the hemocyte characteristics of the Pacific oyster Crassostrea gigas and the carpet shell clam Ruditapes decussatus acclimated 1 or 2 days under emersed conditions at 14 ± 1 °C and for 1, 2, 7, or 10 days to flowing seawater conditions (submerged) at 9 ± 1 °C, when compared to hemolymph withdrawn from organisms sampled in the field and immediately analyzed in the laboratory (unacclimated). The hemocyte characteristics assessed by flow cytometry were the total (THC) and differential hemocyte count, percentage of dead cells, phagocytosis, and reactive oxygen species (ROS) production. Dead hemocytes were lower in oysters acclimated both in emersed and submerged conditions (1%-5%) compared to those sampled in the field (7%). Compared to oysters, the percentage of dead hemocytes was lower in clams (0.4% vs. 1.1%) and showed a tendency to decrease during acclimatization in both emersed and submerged conditions. In comparison to organisms not acclimated, the phagocytosis of hemocytes decreased in both oysters and clams acclimated under submerged conditions, but was similar in those acclimated in emersed conditions. The ROS production remained stable in both oysters and clams acclimated in emersed conditions, whereas in submerged conditions ROS production did not change in both the hyalinocytes and granulocytes of oysters, but increased in clams. In oysters, the THC decreased when they were acclimated 1 and 2 days in submerged conditions and was mainly caused by a decrease in granulocytes, but the decrease in THC in oysters acclimated 2 days in emersed conditions was caused by a decrease in hyalinocytes and small agranular cells. In clams, the THC was significantly lower in comparison to those not acclimated, regardless of the conditions of the acclimatization. These findings demonstrate that hemocyte characteristics were differentially affected in both species by the tested conditions of acclimatization. The phagocytosis and ROS production in clams and phagocytosis in oysters were not different in those acclimated for 1 day under both conditions, i.e. emersed and submerged, and those sampled in the field (unacclimated). The THC was significantly affected by acclimatization conditions, so the differences between clams and oysters should be considered in studies where important concentrations of hemocytes are required. The difference in the immune response between both species could be related to their habitat (epifaunal vs. infaunal) and their ability of resilience to manipulation and adaptation to captivity. Our results suggest that functional characteristics of hemocytes should be analyzed in both oysters and clams during the first 1 or 2 days, preferably acclimated under emersed rather than submerged conditions.

Mechanisms of copper accumulation in isolated mantle cells of the marine clam Mesodesma mactroides.

In vivo copper accumulation was determined in tissues (mantle, gills, digestive gland, and hemolymph) following exposure to Cu (5 µM) for up to 96 h. Mantle was the tissue that accumulated the most Cu, followed by gill, digestive gland, and hemolymph. Therefore, in vitro Cu accumulation was evaluated in isolated mantle cells exposed to 0.5, 1.0, 2.5, and 5.0 µM Cu for 1 and 3 h. After both exposure times, no change in cell viability was observed. However, a significant Cu accumulation was observed in cells exposed to 2.5 and 5.0 µM Cu. Cell exposure to 2.5 µM Cu for 1 h did not affect the ionic (Na(+), K(+), Ca(2+), and Cl(-)) content of isolated mantle cells, characterizing an "ideal" noneffect concentration for the study of the involvement of different ion-transporting proteins (Na(+), K(+), and Cl(-) channels; Na(+)/K(+) 2Cl(-) and Na(+)/Cl(-) cotransporters; Na(+)/Ca(2+), Cl(-)/HCO3-, and Na(+)/H(+) exchangers; Na(+)/K(+) -ATPase; V-ATPase; and carbonic anhydrase) in Cu accumulation. Isolated cells were pre-exposed (30 min) to specific blockers or inhibitors of the ion-transporting proteins and then exposed (1 h) to Cu (2.5 µM) in the presence of the drug. A significant increase of 29.1 and 24.3% in Cu accumulation was observed after cell incubation with acetozalamide (carbonic anhydrase inhibitor) and NPPB (Cl(-) channels blocker), respectively. On the other hand, a significant decrease (48.2%) in Cu accumulation was observed after incubation with furosemide (Na(+) /K(+)/2Cl(-) blocker). Taken together, these findings indicate the mantle as an important route of Cu entry in M. mactroides, pointing to the cotransporter Na(+)/K(+)/2Cl(-) as a major mechanism of Cu accumulation in mantle cells of the clam.

Effect of copper on ion content in isolated mantle cells of the marine clam Mesodesma mactroides.

The effect of copper on ion content (Na(+), K(+), Ca(2+), and Cl(-)) was evaluated in isolated mantle gills of the marine clam Mesodesma mactroides. Clams were collected at the Mar Grosso Beach (São José do Norte, Rio Grande do Sul [RS], southern Brazil), cryoanesthetized, and had their mantles dissected. Mantle cells were isolated and incubated in a calcium-free phosphate solution without (control) or with Cu (CuCl(2)). Cells were exposed to Cu for 1 h (5 µM) or 3 h (2.5 and 5 µM). In cells incubated with 2.5 µM Cu, a significant decrease in intracellular Cl(-) content was observed. However, in cells incubated with 5.0 µM Cu, significant reductions in Na(+), K(+), and Cl(-) intracellular content were observed. Given the mechanisms involved in ion transport in mantle cells of the marine clam M. mactroides, the findings described here suggest that Cu exposure inhibits carbonic anhydrase and Na(+)/K(+) -ATPase activity. Also, it can be suggested that Cu is competing with Na(+) for the same mechanisms of ion transport in the cell membrane, such as the Na(+) channels and the Na(+)/K(+)/2Cl(-) cotransporter. Results from the present study also clearly indicate that processes involved in cellular anion regulation are more sensitive to Cu exposure than those associated with the cellular cation regulation. Characterization of sites for Cu accumulation and toxicity in aquatic animals is important for derivation of metal binding constants at the biotic ligand. Also, identification of the mechanism of metal toxicity is needed for modeling metal accumulation in the biotic ligand and its consequent toxicity. Therefore, the findings reported here are extremely valuable for the development of a biotic ligand model version for marine and estuarine waters.

Comparison of the spermatozoan morphology of Isognomon bicolor and Isognomon alatus (Mollusca, Bivalvia, Isognomonidae).

In this study we used transmission and scanning electron microscopy to examine the spermatozoan structure of Isognomon bicolor and Isognomon alatus. The spermatozoa of both species were of the primitive or ect-aquasperm type. The acrosomal morphologies were essentially similar but the top of the acrosomal vesicle in I. bicolor sperm had a slightly flattened edge whereas the apex of the acrosomal vesicle of I. alatus sperm had a rounded outline. This difference suggested that acrosomal morphology could be an important character for taxonomic differentiation. In the present work, the results demonstrated that the gamete ultrastructure of the two distinct species I. alatus, from Panama, and I. bicolor, from the southeastern region of Brazil, were similar to the other studied species of the superfamily Pterioidea.

Mast cells are present in epithelial layers of different tissues of the mollusc Anomalocardia brasiliana. In situ characterization of heparin and a correlation of heparin and histamine concentration.

Heparin, other sulphated glycosaminoglycans and histamine were extracted from various dissected organs of Anomalocardia brasiliana, a mollusc from the South Atlantic, and quantified. A good correlation between heparin and histamine content was found in the labial palp, intestine, ctenidium, mantle and foot tissues. The tissue location of metachromatic cells, putatively containing heparin, was identified histologically with Alcian Blue, Toluidine Blue, Masson trichrome, Haematoxylin-Eosin and PAS. Except for the foot, cells containing metachromatic granules were found in the epithelium surfaces of all the organs analysed. An in situ identification of heparin using nitrous acid and heparinase degradation has established unequivocally the presence of this compound in the metachromatic cells. The location of 'mast-like' cells at the epithelium surface of mollusc tissues exposed to the environment are very similar to the distribution of mammalian and other vertebrate mast cells and gives support to the suggestion for a role of mast cells in defense mechanisms.