Mantle Tissue Research Articles

Regeneration of excised mantle tissue by the silver-lip pearl oyster, Pinctada maxima (Jameson)

This study investigated the capacity for mantle regeneration in the pearl oyster Pinctada maxima. Oysters were anaesthetised with 2.5 mL L −1 prophylene phenoxetol prior to a piece of tissue (approximately 10 × 30 mm 2) being excised from the ventral region of the mantle. In the first experiment, 56 oysters with mean (±SD) dorso–ventral measurement (DVM) of 125.5 ± 8.9 mm had tissue excised from either the right mantle lobe, left mantle lobe or both mantle lobes. Following a further three month period in suspended culture, oyster survival was recorded and two oysters were selected arbitrarily from each group to be sacrificed for histological examination of healed mantle. In the second experiment 36 oysters with mean (±SD) DVM of 151.6 ± 13.4 mm were used for excision of the distal part of the ventral region of the left mantle lobe. Two oysters were sampled at 1, 3, 6, 12, 24, 36, 48, 72 and 120 h (5 days) after mantle excision, and then at 12, 24, 45, 72 and 90 d after mantle excision for histological and histochemical analysis of mantle regeneration. There was almost 100 percent survival in both experiments. Healing and regeneration of mantle tissue in oysters subject to excision from the left, right or both mantle lobes was evident, with regenerated mantle appearing similar to non-regenerated mantle. All external and internal components of non-regenerated mantle were present in regenerated mantle tissue. Epithelization signifying wound healing occurred within 36–72 h and was characterised by a reduced wound area, haemocyte infiltration and accumulation, and cell dedifferentiation. Within 48 h of mantle excision, the latero-ventral edges of the wound flexed dorsally and attached to the dorsal edge of the wound reducing the wound area. Between five and twelve days after excision, the distal part of the mantle had divided into three small lobes which developed into the outer, middle and inner mantle folds two weeks later. Ninety days after excision the mantle had completely regenerated with histological observations indicating no difference in epithelial structure or in other internal mantle accessories when compared to non-regenerated mantle. Shell material began to be secreted onto the shell by regenerating mantle twelve days after excision. Initially this occurred in a position dorsal to the non-injured mantle edge. However, forty-five days after mantle excision, regenerated mantle had extended ventrally to a position similar to that of non-injured mantle. Nacre deposition by regenerated mantle had now reached the same position ventrally as that of non-injured mantle indicating full acquisition of nacre secreting abilities by regenerated mantle.

Histopathologic biomarker response of clam, Ruditapes decussates, to organophosphorous pesticides reldan and roundup: A laboratory study

Clam Ruditapes decussates was used as a biomonitoring organism to measure the potential impact of pollution. The present study investigated histological alterations of R. decussates following a long-term exposure to a sublethal-level of pesticides Reldan and Roundup. The clams were taken to the laboratory and kept for days to adjust to a continuous closed flow system with quality water control. After the adjustment period, clams were exposed either to sublethal concentrations of Reldan (0.6 μl L−1) or Roundup (1.1 μl L−1) for 90 days. Clams were taken to determine the histological damage at 30, 60 and 90 days. Most histological alterations were highly localized in the glandular cells of the gills, mantle tissues and digestive gland; secretions of glandular cells were found to be increased by increasing the time of exposure. Histopathological results showed the activation of resistance mechanisms that allowed the clams to survive under sublethal stresses. Thus, histological changes on localized tissues were sensitive and they have positive correlation to the time of exposure to the pesticides, suggesting that they may serve as biomarkers for Reldan and Roundup exposures.

Purification and characterization of a new Ca2+-dependent protein kinase C in mussel (Mytilus galloprovincialis Lmk.) mantle

An enzyme that can be included into the so-called conventional PKCs has been purified to homogeneity from the mantle tissue of the sea mussel Mytilus galloprovincialis. This enzyme has a molecular weight of 60 kDa, which is DAG-dependent, PS-activated, and Ca2+-dependent. It was separated from a Ca2+-independent PKC (p105) (Mercado et al., Mol Cell Biochem 233:99-105, 2002) by means of an ionic exchange chromatography on DE-52 cellulose. The molecular weights and kinetic properties of both the enzymes are different. The protein p60 is broadly distributed among the tissues, which suggests that it may carry out specific functions, different from those performed by p105.

Mantle-to-shell CaCO3 transfer during shell repair at different hydrostatic pressures in the deep-sea vent mussel Bathymodiolus azoricus (Bivalvia: Mytilidae)

Calcium carbonate transfer was experimentally examined in hydrothermal mussels Bathymodiolus azoricus, which were collected from 850 m depth at Menez Gwen hydrothermal vent site (31°31′W, 37°50′N) on the Mid Atlantic Ridge in May 2007. In each of four 10-day experiments, groups of mussels were maintained at atmospheric pressure or re-pressurised to depths relevant to their site of occurrence, i.e. 850 m depth at Menez Gwen, 1,750 m at Lucky Strike (31°31′W, 37°18′N) and 2,300 m at Rainbow (31°31′W, 36°13′N). The shells of experimental mussels were perforated and mantle tissue was fixed for light and TEM studies at days 7 and 10 following the injury. Simultaneously, haemocytes from the extrapallial fluid (EPF) at the site of induction were studied. At day 7 the response was most intense in the middle fold of the mantle margin and possibly proportional to hydrostatic pressure. At day 10 the epithelial cells on the mantle surface facing the body cavity produced copious organic secretions that avidly bound calcium. Haemocyte migration was noted within the mantle tissue, and the haemocytes at the mantle surface facing the shell had a Ca-positive granular content. Large haemocytes were detected in the EPF at the injury site, and some showed evidence of an immune reaction while others showed Ca-positive granular content. These results suggest that haemocytes are involved in shell repair in these deep-sea mussels just as in some freshwater and shallow marine molluscs.

Toxicity of lindane (γ-hexachloroxiclohexane) in Sparus aurata, Crassostrea angulata and Scrobicularia plana

The purpose of the present study was to research the sublethal and/or lethal effects produced by the exposure of fish and shellfish to the γ isomer of lindane, γ-hexachlorocyclohexane (γ-HCH). The teleostean fish Sparus aurata and the shellfish Crassostrea angulata and Scrobicularia plana, were exposed to 16 μ g/L of lindane for 15 days. Samples of different fish (liver, kidney and gills) and shellfish (gills, gut, digestive gland and mantle) tissues were extracted and processed for histopathological observations. Although mortality was not detected during the bioassay, sublethal effects (histopathological alterations) were observed. Vacuolization in the liver cells and lamellar fusion in gills from exposed fish were observed. Disorganization of normal gill structure, epithelial desquamation with the disappearance of apical ciliature in intestine, and inflammatory response in mantle from exposed shellfish were also observed. Thus, it can be concluded that the lindane concentration employed in the present research did not produce lethal effects in the exposed organisms but it caused sublethal effects. Lindane has time-dependent multiple toxic effects in S. aurata, C. angulata and S. plana, which were more severe at the end of the experimental time. The toxicological implications arising from these results are subjects for further multiconcentration tests dealing with lethal responses (mortality) or with sublethal responses (cellular/molecular biomarkers) of the aforementioned species.

Histological localization and analytical evaluation of heparin in the wedge clam Donax Cuneatus (Linnaeus, 1758)

Glycosaminoglycan (GAG) of the heparin family was localized in the tissues of the bivalve mollusk Donax cuneatus. Different histological staining procedures such as Alcian blue, azure-A, safranin and periodic acid-Schiff reagent (PAS) were followed to test the presence of GAG in various tissues of this bivalve mollusk. GAG was found in the tissues of gill, palp, ctenidia, foot, mantle, inhalant and adductor muscle in order of decreasing importance. Palp, gill and ctenidia tissues showed high staining intensity with PAS (94.8%) processing and Alcian blue (92.4%) indicated the presence of sulfated polysaccharide. Crude GAG was isolated from these tissues using the cetylpyridinium chloride precipitation method. The product obtained was tested for its anticoagulant property along with standard heparin, which was partially purified by fractionation by anion exchange chromatography using a diethylaminoethyl (DEAE)-cellulose column. The isolated GAG and fractionated GAG were also compared with standard heparin through Fourier transform infrared (FT-IR) to find relevant functional groups. The bivalve GAG was subjected to DEAE-cellulose fractionation for further purification and its chemical components were analyzed. The subsequent comparison with commercial heparin showed that D. cuneatus could be a potent source of heparin for commercial use.

Reproductive system and the spermatophoric reaction of the mesopelagic squid Octopoteuthis sicula (Rüppell 1844) (Cephalopoda: Octopoteuthidae) from southern African waters

Reproductive features of the poorly known oceanic squid Octopoteuthis sicula are described and quantified to gain insight into the reproductive biology of the species. The data are based on 39 complete and partial specimens from southern African waters, collected between 1975 and 2005. The specimens ranged in mantle length from 38 mm to 290 mm and included juveniles and mature females and males. The species shows female-biased sexual size dimorphism. Ovulation is asynchronous, indicating a repeated spawning strategy. Males transfer spermatophores presumably by using their long terminal organ. Spermatangia (discharged spermatophores) were found implanted in several parts of the body in both females and males, including in the anterior dorsal and ventral rugose, semi-gelatinous mantle tissue of maturing and mature females. This modified mantle tissue was only well developed in females. The morphologies of the spermatophore and the spermatangium of O. sicula are described. The spermatophoric reaction is reconstructed, using various stages of discharge, to provide insight into the process of intradermal implantation of spermatangia of this species.

Identification and expression of TRAF6 (TNF receptor-associated factor 6) gene in Zhikong Scallop Chlamys farreri

Tumor necrosis factor receptor-associated factor 6 (TRAF6), a key signaling adaptor molecule common to the TNFR superfamily and IL-1R/TLR family, is important not only for a diverse array of physiological processes functions of the TNFR superfamily, but also is involved in adaptive immunity and innate immunity. In this report, the first bivalve TRAF6 (named as CfTRAF6) gene is identified and characterized from Zhikong scallop Chlamys farreri. The full-length cDNA of CfTRAF6 is of 2510 bp, consisting of a 5′-terminal untranslated region (UTR) of 337 bp, a 3′-terminal UTR of 208 bp with a canonical polyadenylation signal sequence AATAAA and a poly (A) tail, and an open reading frame (ORF) encoding a polypeptide of 655 amino acids. The predicted amino acid sequence of CfTRAF6 comprises characteristic motifs of the TRAF proteins, including a Zinc finger of RING-type, two Zinc fingers of TRAF-type, a coiled-coil region, and a MATH (the meprin and TRAF homology) domain. The overall amino acid sequence identity between CfTRAF6 and other TRAF6s is 28–68%. Phylogenetic analyses of CfTRAF6 sequence with TRAF sequences from other organisms indicate that CfTRAF6 is a true TRAF6 orthologue. The mRNA expression of CfTRAF6 in various tissues is measured by Real-time RT-PCR. The mRNA transcripts are constitutively expressed in tissues of haemocyte, muscle, mantle, heart, gonad and gill, but the highest expression is observed in the gonad. The temporal expressions of CfTRAF6 mRNA in the mixed primary cultured haemocytes are recorded after treatment with 20 μg mL −1 and 0.5 μg mL −1 peptidoglycan (PGN). The expression level of CfTRAF mRNA is down-regulated from 1.5 h to 3 h after the treatment with 0.5 μg mL −1 PGN, and then recovers to the original level. While the expression of CfTRAF6 is obviously decreased after treatment with 20 μg mL −1 PGN, and reach the lowest point (only about 1/9 times to control) at 3 h. The result suggests that CfTRAF6 can be greatly regulated by PGN and it may be involved in signal transduction and immune response of scallop.

Modulatory effects of hard clam ( Mercenaria mercenaria) tissue extracts on the in vitro growth of its pathogen QPX

Quahog parasite unknown (QPX) is a fatal protistan parasite affecting cultured and wild hard clams Mercenaria mercenaria along the northeastern coasts of the USA and maritime Canada. Field investigations and laboratory transmission studies revealed some variations in the susceptibility of different hard clam stocks to QPX infection. In this study, we used in vitro QPX cultures to investigate the effect of plasma and tissue extracts from two different clam stocks on parasite survival and growth. Results demonstrated the presence of factors in clams that significantly modulate QPX growth. Extracts from gills and mantle tissues as well as plasma inhibited in vitro QPX growth, whereas foot and adductor muscle extracts enhanced parasite growth. Investigations of anti-QPX activities in plasma from two clam stocks displaying different susceptibility toward QPX disease in vivo demonstrated higher inhibition of QPX growth by plasma from New York (resistant) clams compared to Florida (susceptible) clams. Some clams appeared to be deficient in inhibitory factors, suggesting that such animals may become more easily infected by the parasite.

Cloning of a cDNA encoding the tail region of non-muscle myosin II from the mantle tissue of scallopPatinopecten yessoensis

Cloning of a cDNA encoding the tail region of non-muscle myosin II from the mantle tissue of scallop<i>Patinopecten yessoensis</i>

Phylogenetic and morphological characterisation of the green algae infesting blue musselMytilus edulisin the North and South Atlantic oceans

Blue mussels Mytilus edulis with shell deformations and green pustules containing parasitic algae were collected at 3 coastal sites (Burøy, Norway; Bockholm, Denmark; Goose Green, Falkland Islands). A comparative study, including mussel histopathology, algal morphology, ultrastructure and phylogenetic position was performed. Green pustules were mainly located in the posterior portion of the mantle and gonad tissues and the posterior adductor muscle. Electron microscopy confirmed the presence of algal cells with similar morphology to Coccomyxa parasitica. Algae were oval shaped with a single nucleus and chloroplast, 1 or 2 mitochondria and a dense granular cytoplasm with a lipid inclusion body, Golgi apparatus and small vesicles. Partial small subunit (SSU) rRNA phylogeny confirmed the inclusion of parasitic algae into the Coccomyxa clade. However, the sequence identity between almost full SSU rRNA sequences of parasitic algae and others in this clade yielded an unexpected result. Green algae from mussels were distant from C. parasitica Culture Collection of Algae and Protozoa (CCAP) strain 216/18 (94% identity), but very similar (99% identity) to C. glaronensis (a lichen endosymbiont) and green endophytes from the tree Ginkgo biloba. The CCAP strain 216/18 was a sister sequence to Nannochloris algae, far from the Coccomyxa clade. These results suggest a misidentification or outgrowth of the original CCAP strain 216/18 by a different 'Nannochloris-like' trebouxiophycean organism. In contrast, our sequences directly obtained from infested mussels could represent the true C. parasitica responsible for the green pustules in blue mussels.

Monthly variation of condition index, energy reserves and antibacterial activity in Pacific oysters, Crassostrea gigas, in Stansbury (South Australia)

This paper investigates the temporal responses of 2-year old Crassostrea gigas to environmental changes in Stansbury, South Australia from September 2005 to October 2006. A total of 360 oysters were grown in one-line baskets on the farm using six replicates that were sampled monthly. A range of environmental parameters were assessed and correlated against biological indicators for oyster condition, metabolism and antimicrobial activity. Food availability by chlorophyll a, was low throughout the study period (0.5–1.5 µg L − 1 ) and was significantly correlated to phosphorus concentrations. The condition index and shell weight of oysters significantly increased over the year, with the condition index dropping after spawning but then recovering within one month. Significant temporal variation in energy storage and utilization were observed in different tissues over the year. Glycogen in the mantle tissue was influenced by reproduction and correlated to chlorophyll a levels, but not in the gill or adductor muscle. The mantle glycogen and gill protein reached the lowest level in February when spawning occurred and presented evidence for seasonal variation in oyster metabolic activity. However, mantle and adductor muscle proteins did not drop after spawning indicating that these proteins contribute little to gametogenesis. Hemolymph protein was negatively correlated to water temperature and chlorophyll a, reaching the lowest level during summer. Hemolymph antibacterial activity significantly decreased after spawning, implying that the period of post-spawning is critical for oyster health. This study revealed trade-offs in the energy budget between immune resistance, growth, and reproduction. The results indicate that in a lean water environment, spawning events significantly regulate metabolic and immune capacities of oysters and a second year of rearing increased meat and shell weight but not the shell length. These findings are applicable to the management and development of oyster aquaculture within temperate southern hemisphere.

Identification of multiple isoforms of the cAMP‐dependent protein kinase catalytic subunit in the bivalve mollusc Mytilus galloprovincialis

Several isoforms of the cAMP-dependent protein kinase catalytic subunit (C-subunit) were separated from the posterior adductor muscle and the mantle tissues of the sea mussel Mytilus galloprovincialis by cation exchange chromatography, and identified by: (a) protein kinase activity; (b) antibody recognition; and (c) peptide mass fingerprinting. Some of the isozymes seemed to be tissue-specific, and all them were phosphorylated at serine and threonine residues and showed slight but significant differences in their apparent molecular mass values, which ranged from 41.3 to 44.5 kDa. The results from the MS analysis suggest that at least some of the mussel C-subunit isoforms arise as a result of alternative splicing events. Furthermore, several peptide sequences from mussel C-subunits, determined by de novo sequencing, showed a high degree of homology with the mammalian Calpha-isoform, and contained some structural motifs that are essential for catalytic function. On the other hand, no significant differences were observed in the kinetic parameters of C-subunit isoforms, determined by using synthetic peptides as substrate and inhibitor. However, the C-subunit isoforms separated from the mantle tissue differed in their ability to phosphorylate in vitro some proteins present in a mantle extract.

Shell layer variation in trace element concentration for the freshwater bivalve Elliptio complanata

While the elemental chemistry of marine mollusk shells contains valuable environmental information, extending these interpretations to freshwater bivalve shells may be problematic, given the wide range of aqueous chemistries that exist in freshwater environments. To better understand the significance of these records, 20 bivalves were collected from four freshwater streams to determine the geochemical relationships that exist between the bivalve shell and their sources streams. The concentrations of manganese (Mn), copper (Cu), strontium (Sr), barium (Ba), and calcium (Ca) were analyzed by laser ablation inductively coupled plasma mass spectrometry across the inner (INL) and outer nacreous layer (ONL) of each shell for comparison to stream data collected over the period of shell growth. Within an individual shell, the content of Mn, Sr, and Ba were significantly higher in the INL than the ONL, while Cu concentrations were similar. Strontium and Ba concentrations co-varied between the two layers in 1:1 relationships, while Mn displayed a preferential enrichment in the INL that increased from 2:1 to 5:1 as the Mn content of the shell decreased. Each elemental profile can be correlated between shell layers, except for the trace element Cu, which appeared to be more closely related to the organic content of the shell. These results suggest that the major element geochemistry of the shell layers differentially reflect the aqueous chemistry of the water in which a bivalve lived, but that these records are most likely overprinted by physiological processes specific to the mantle tissue from which a particular shell layer is secreted. Distribution coefficients (shell M /Ca:water M /Ca, where M = trace element of interest) were calculated using the median molar elemental concentration for each shell (by layer), and the appropriate water concentration. Bivalves from a contaminated site were excluded from this analysis because their shells were anomalous in size and color. For the remaining 15 shells, distribution coefficients (INL and ONL) were: Mn (0.50 and 0.21), Sr (0.26 and 0.17), and Ba (0.05: INL only). Inner nacreous layer values were comparable to the upper end of published estimates for freshwater bivalves and fish otoliths, while the ONL values were comparable to the lower range of values. Inclusion of shells from the contaminated site resulted in the calculation of distribution coefficients that fell outside the range of published estimates. These results suggest that exposure to pollutants may have a bearing on the biological factors that control the elemental concentration of bivalve shells in freshwater environments. While researchers generally avoid sampling shell material from the INL because of the potential for shell dissolution, the similarity in elemental profile trends observed here suggests that both the INL and ONL record accurate geochemical information, but in distinct forms. An obvious advantage of INL analysis is the relative increase in elemental concentration, but this is afforded at the expense of spatial resolution. Considered collectively, these results suggest that valuable information can be gleaned from the elemental concentration of freshwater bivalve shells when care is taken in the choice of material from which these records are extracted.

Polycyclic aromatic hydrocarbons in surface seawater and in indigenous mussels ( Mytilus galloprovincialis) from coastal areas of the Saronikos Gulf (Greece)

Polycyclic aromatic hydrocarbons (PAHs) were identified and measured in surface seawater and in the tissues (gills and mantle) of indigenous black mussels, Mytilus galloprovincialis, collected from three coastal sites of Saronikos Gulf (Greece), a gulf that exhibits high levels of pollution. The total PAHs measured by spectrofluorometry in the surface seawater were found in the range of 425–459 ng L −1 at the most polluted sites 1 and 2 (Elefsis Bay–Salamis Island) and in the range of 103–124 ng L −1 at site 3 (Aegina Island). PAHs' sources in seawater were identified by application of specific PAH ratios, such as phenanthrene/anthracene and fluoranthene/pyrene. Levels of PAHs in soft tissues (gills and mantle) of indigenous mussels were much higher than those reported for seawater. Total PAH concentrations in mantle tissues were in the range of 1300–1800 ng g −1 dry weight (dw) tissue at sites 1 and 2 and approximately 380 ng g −1 dw at site 3. In gill tissues total PAH concentrations were in the range of 1480–2400 ng g −1 dw at sites 1 and 2 and approximately 430 ng g −1 dw at site 3. PAHs composition was dominated by two-, three- and four-ring compounds in seawater, where 17 different PAH compounds were identified and measured in mussel tissues. Mussels can be used as sentinel organisms to monitoring PAHs' contamination, since they concentrate PAHs from the surrounding water media and therefore making the chemical analysis simpler and less prone to error than that for water. In surface seawater possible weathering and photodegradation due to hot climates contribute to reduced PAHs concentrations.

Culture of outer epithelial cells from mantle tissue to study shell matrix protein secretion for biomineralization

Mantle tissue plays an important role in shell biomineralization by secreting matrix proteins for shell formation. However, the mechanism by which it regulates matrix protein secretion is poorly understood, largely because of the lack of cellular tools for in vitro study and techniques to evaluate matrix protein secretion. We have isolated the outer epithelial cells of the mantle of the pearl oyster, Pinctada fucata, and evaluated cellular metabolism by measuring the secretion of the matrix protein, nacrein. A novel sensitive sandwich enzyme-linked immunosorbent assay (ELISA) was established to quantify nacrein. Mantle explant culture was demonstrated to provide dissociated tissue cells with high viability. Single dissociated cell types from explant culture were separated by density in a discontinuous Percoll gradient. The outer epithelial cells were isolated from other cell types by their higher density and identified by immunolabeling and ultrastructure analysis. ELISA assays revealed that the outer epithelial cells retained the ability to secrete nacrein in vitro. Moreover, increased nacrein secretion resulted from an increased Ca(2+) concentration in the culture media of the outer epithelial cells, in a concentration-dependent manner. These results confirm that outer epithelial cell culture and the ELISA method are useful tools for studying the regulatory mechanisms of shell biomineralization.

Highly divergent duplicate mannose-6-phosphate isomerase ( Mpi) genes in the blue mussel, Mytilus edulis

Gene duplication has been hypothesized to play a major role in the evolution of genes and genomes and in generating phenotypic diversity among the proteins those genes encode. We have identified duplicate genes for the glycolytic enzyme mannose-6-phosphate isomerase (MPI: EC 5.3.1.8) in marine mussels in the genus Mytilus. Overall, there was only 52% sequence identity (72% similarity) between the proteins encoded by these two genes, designated as Mpi-A and Mpi-B. Based on a comparison of the rate of non-synonymous substitution between orthologous and paralogous Mpi coding sequences obtained from Mytilus edulis and the congener M. trossulus we estimate that the duplication of Mpi in mussels occurred ~ 170 MYA. We detected paralog-specific differences in the ratio of non-synonymous to synonymous substitutions ( k a/ k s) and in the predicted net charge for MPI-A and MPI-B. Using a real-time quantitative RT-PCR assay we observed substantial changes in Mpi-A and Mpi-B transcript levels between tissue types; the strongest expression of Mpi-A was observed in mantle tissue while Mpi-B expression exceeded that of Mpi-A in gill and hepatopancreas tissues. Taken together, these observations suggest that different functional roles have evolved for these two Mytilus Mpi genes subsequent to gene duplication.

Assessment of European cuttlefish ( Sepia officinalis, L.) nutritional value and freshness under ice storage using a developed Quality Index Method (QIM) and biochemical methods

The aim of this study was to determine the nutritional value of adult commercial cuttlefish, to develop a Quality Index Method (QIM) scheme, and to evaluate the application of some biochemical methods commonly used for freshness assessment of fish under ice storage. Additionally, shelf-life was to be determined based on both QIM and suitable biochemical methods. The nutritional value of the cuttlefish mantle in the first 24 h and after 13 days was determined. Captured cuttlefish was composed (g/100 g) by 16.60 ± 0.10 g protein, 0.09 ± 0.01 g fat, 79.55 ± 0.14 g moisture and 1.39 ± 0.03 g of ash. After 13 days of ice storage, cuttlefish was composed (g/100 g) of 11.90 ± 0.28 g protein, 0.17 ± 0.09 g fat, 87.04 ± 0.13 g moisture and 0.52 ± 0.01 g of ash. Differences ( p < 0.001) were found in protein, ash and moisture but not in fat ( p > 0.05). These results seem to indicate that there is impregnation of the iced water into the mantle tissue promoting protein leaching with the melting ice. TVB-N and TMA-N displayed a similar increasing tendency, peaking beyond EEC regulations proposed maximum between the 9th and 10th days. The developed QIM scheme for cuttlefish was composed of 29 demerit points, divided into 4 attributes and 13 parameters. The calculated quality index (QI) evolved linearly with storage time in ice (QI = 2.68 × days in ice − 0.61, R 2 = 0.9866). Storage time could be estimated with an accuracy of ± 1 day, if five cuttlefish from each sample were included in the QIM assessment. The shelf-life was determined as 8 ± 1 days by both type of methods (QIM and biochemical). However, the suitability of some biochemical methods to assess freshness need to be more thoroughly researched.

Energy Storage and Allocation During Reproduction of Pacific Winged Pearl Oyster Pteria sterna at Bahía de la Paz, Baja California Sur, Mexico

Seasonal variations in storage, partitioning, and allocation of energy reserves (proteins, carbohydrates, lipids, and triglycerides) between germinal and somatic tissues (gonad, digestive gland, mantle tissue, and adductor muscle), were investigated related to reproduction of Pacific winged pearl oyster Pteria sterna. Tissue samples were collected every three months and analyzed with histological and biochemical techniques. Energy coefficients were also calculated with data from chemical composition of tissues. Gonad samples in almost all developmental stages occurred throughout the year, suggesting that P. sterna is a multispawning species. The evidence indicates that the main reproductive season runs from January through April (21°C to 22°C) and was identified by higher frequency of ripe gonads, more and larger postvitellogenic oocytes and higher protein, lipid, and triglyceride levels in gonad tissue. Within this study period, there were two spawning peaks, July 2003 and January 2004. Gametogenesis was sustained from energy mainly obtained from the digestive gland and secondly from the adductor muscle. Only proteins from these two tissues were mobilized to the gonad for maturation of sex organs, because carbohydrates were stored despite the progress of gametogenesis. The role of mantle tissue was negligible. P. sterna appears to use a combination of stored reserves (conservative strategy) and food supply (opportunistic strategy) as an overall strategy to regulate reproduction.

Characterization of Calcium Deposition and Shell Matrix Protein Secretion in Primary Mantle Tissue Culture from the Marine Pearl Oyster Pinctada fucata

In this study, we established and characterized a long-term primary mantle tissue culture from the marine pearl oyster Pinctada fucata for in vitro investigation of nacre biomineralization. In this culture system, the viability of mantle tissue cells lasted up to 2 months. The tissue cells were demonstrated to express nacre matrix proteins by RT-PCR, and a soluble shell matrix protein, nacrein, was detected in the culture medium by Western blot analysis. On the other hand, 15 days after initiating culture, a large amount of calcium deposits with major elements, including calcium, carbon, and oxygen, were generated in the mantle explants and cell outgrowth area. The quantity and size of calcium deposits increased with the prolonged cultivation, and their location and nanogranular structure suggested their biogenic origin. These calcium deposits specifically appeared in mantle tissue cultures, but not in heart tissue cultures. Taken together, these results demonstrate that the mantle tissue culture functions similarly to mantle cells in vivo. This study provides a reliable approach for the further investigation on nacre biomineralization at the cellular level.