Quantitative studies on the reproduction of black scallop,Chlamys varia (L.) from lanveoc area (Bay of Brest)

The values of gonad index calculated by the allometric relationships of dry gonad weight over dry body weight of the black scallop, Chlamys varia (L.), gave peak values for May and September during 1976 and 1977. Histological studies on gonads from natural populations and periodic biopsies of gonads from marked animals in experimental cages showed that, in addition to intermittent spawnings between May and September, two peak spawning periods were noteworthy. These were a partial spawning in May–June and a complete spawning in September–October. The energy spent as gametes for these two spawnings has been calculated for individual males and females in each age group of the population and the cost of spawning by the whole population has been estimated. The population spent more energy for spawning in 1977 (144.49 kcal/100 m²) than in 1976 (83.70 kcal/100 m²) and the reproductive effort of an average adult was 0.04 in 1976 and 0.10 in 1977.     

Growth and biannual recruitment of the black scallop Chlamys varia (L.) in lanveoc area,Bay of Brest

Data on growth of the black scallop Chlamys varia (L.) were obtained by taggine, raft cage rearing, reading winter growth rings, and analysis of length-frequency distributions. Von Bertalanffy growth curves were fitted to each set of data by least-squares regression techniques involving no transformation of variates. The results obtained by the different methods are comparable except for those obtained by length-frequency distribution analysis. Length frequency distribution analysis seems to be complicated by the presence of a biannual recruitment. Counting growth rings seems to be a more accurate and easier method for growth estimates. The sizes at rings are different for individuals born in spring and for those born in autumn; the growth curves derived for these two types of animals remain different throughout their whole life. The relative proportions of individuals originating from each of the two recruitment groups have remained remarkably homogeneous for the last five years. Comparative study of the allometric relationships between length and width of the shell of the animals from the two recruitment groups showed no significant difference.     

Characterization of a myostatin-like gene from the bay scallop, Argopecten irradians

A complete cDNA was cloned from the bay scallop (Argopecten irradians) that codes for a 382-amino-acid myostatin-like protein (sMSTN). The sMSTN sequence is most similar to mammalian myostatin (MSTN), containing a conserved proteolytic cleavage site (RXXR) and conserved cysteine residues in the C-terminus. Based on quantitative RT-PCR, the sMSTN gene is predominantly expressed in the adductor muscle, with limited expression in other tissues. Using the sMSTN sequence, a Ciona MSTN-like gene was also identified from the Ciona intestinalis genome. These findings indicate that the MSTN gene has been conserved throughout evolution and suggests that MSTN could play a major role in muscle growth and development in invertebrates, as it does in mammals.     

Characterisation of monoclonal antibodies to haemocyte types of scallop (Chlamys farreri)

Four monoclonal antibodies (MAbs) (1E7, 1F12, 2H5, 2C6) against haemocytes of scallop (Chlamys farreri) were produced by immunising Balb/C mice. Analysed by the indirect immunofluorescence assay test (IIFAT), immunocytochemical assay, flow cytometry (FCM) and Western-blotting, they showed specificity for more than one haemocyte type (hyalinocyte and granulocyte) and various haemocyte components of scallop. Using IIFAT to detect monolayers separated from gradient density centrifugation, the four MAbs were positive with haemocytes at different interfaces. The percentage of positive cells (percent reactivity, PR) that MAb 1E7 reacted with at the 20-30%, 30-40% and 40-50% interfaces were 43.50%, 41.25% and 60.00% respectively, PR of MAb 1F12 were 31.00%, 63.50% and 41.00%, MAb 2C6 were 11.00%, 51.00%, 77.00%, and MAb 2H5 were 20.25%, 34.75%, 38.25%. For the immunocytochemical assay, MAb 1E7 1F12 and 2H5 was positive with the cytoplasm of both hyalinocyte and granulocyte, 2C6 was positive with the membrane and cytoplasm of hyalinocyte and granulocyte. Analysed by FCM, the PR of the four MAbs (1E7, 1F12, 2H5, 2C6) with haemocytes were 54.23%, 38.56%, 56.4%, and 79.7% respectively; moreover, the PR with different haemocyte types was variable. The results of Western-blotting showed that MAb 1E7 recognised an antigen of molecular weight 200 kDa, MAb 2C6 an antigen of 60 kDa, however, MAb 1F12 reacted with antigens of 70 kDa, 60 kDa and 45 kDa. There was no protein band that MAb 2H5 detected. In conclusion, 2C6 seems to be a very promising MAb to identify and differentiate granulocytes, and the four MAbs will be used in further studies on cellular defence mechanism research.     

Purification, properties and temperature dependence of the adenosine deaminase from a poikilotherm (bay scallop)

The adenosine deaminase of the digestive diverticulum of the bay scallop was purified and electrophoresis of the purified enzyme yielded a single enzymatically active band at several different pH values. A molecular weight of 130,000 was estimated using gel filtration and sucrose density gradient centrifugation. The enzyme had spectral properties typical of simple proteins and its isoelectric point proved to be 4.8. The scallop enzyme was stable at room temperature from pH 5.0 to 7.0, and in this range it was exceptionally resistant to heat inactivation.The effect of the substrate, adenosine, on the reaction velocity was followed over a 10,000-fold concentration range, and no deviation from Michaelis-Menten kinetics was observed. The following rate equation applies to the enzyme: $\text{1}\text{^v}\text{= (}\text{1}\text{α[}\text{S}\text{]}\text{) + (}\text{1}\text{β}\text{)}$.The effect of pH on the reaction, using adenosine as the substrate, was studied; and it was found that pH had a much greater effect on the α parameter of the rate equation than on the β parameter and that pH had little effect on the apparent activation energy of either parameter. The apparent activation energy of the β parameter was 12.2 kcal with adenosine as the substrate, while the apparent activation energy of the α parameter was zero. The α parameter of the rate equation, using other substrates, was also insensitive to temperature.     

Deoxyribonuclease II from the Icelandic scallop (Chlamys islandica): isolation and partial characterization

A deoxyribonuclease (DNase) was isolated from viscera of the cold-adapted marine bivalve Icelandic scallop. The 42 kDa DNase was shown to be a single polypeptide which catalyses DNA hydrolysis in the absence of divalent cations. The isolated enzyme showed maximal activity at pH 6 and no activity above pH 7.2 against native DNA. The scallop DNase was slightly more susceptible to heat denaturation than porcine DNase II and makes double-strand breaks in circular DNA substrate as the porcine enzyme. The N-terminal sequence of the scallop DNase was shown to be closely similar to DNase II (EC 3.1.22.1) proteins from other organisms. The scallop DNase is in addition to plancitoxin I from A. planci, the only DNase II enzyme isolated from marine invertebrates.     

Morphological and chromosomal variation of the Dryopteris varia (L.) Kuntze complex (Dryopteridaceae) in Korea

The Dryopteris varia complex is highly variable in morphology, resulting in taxonomic confusion in delimiting taxon boundaries and determining relationships. We have examined the variation in morphology, chromosome number and mode of reproduction of the Korean members of the D. varia complex to clarify their taxonomic identities. Landmark analysis of the leaf blades and pinnae and the principal components analysis of 31 morphological characters revealed seven entities within the D. varia complex in Korea; these comprise D. varia s. str., D. pacifica, D. sacrosancta, D. bissetiana, D. saxifraga, D. saxifragivaria, and the Suak population which is considered to be a new taxon. Mitotic chromosome counts and examination of reproduction modes indicated that D. bissetiana in Korea appears to be agamosporous with diploid (2n = 82) or triploid (2n = 123) chromosome numbers. However, D. saxifraga is sexual diploid or agamosporous triploid, and the other taxa are agamosporous triploid. Dryopteris bissetiana, D. saxifraga and D. saxifragivaria are similar in major morphological characteristics, but show differences in attachment and shape of rachis scales. The results also suggest that agamosporous triploid D. saxifragivaria was probably derived from hybridization between sexual diploid D. saxifraga and D. bissetiana.     

The cardiac responses of the scallop Pecten maximus (L.) to respiratory stress

Heart activity of Pecten maximus (L.) has been recorded during various forms of experimentally induced respiratory stress. There was considerable variation in the responses of individual scallops but bradycardia generally occurred in response to all forms of respiratory stress, with the rate of fall in heart rate dependent upon the severity of hypoxia.When oxygen tension declined slowly in a closed respirometer there was regulation of both heart rate and oxygen consumption. The critical tension, P_c, for oxygen consumption lay between 70 and 80 mm Hg, and corresponded with a slight regulatory upswing of the heart rate, whereas the P_c for heart rate was much lower at 20–30 mm Hg. Sudden transfer to deoxygenated water for 3 h resulted in very rapid bradycardia and there was a rapid recovery and initial overshoot of the normal rate on return to well-oxygenated sea water. Aerial exposure for 3 h produced more gradual bradycardia followed by gradual recovery on return to sea water.The results of this work are compared in some detail with previous work on other species of bivalve from different geographical areas and habitats, and the mechanisms controlling cardiac and respiratory regulation are discussed. It is concluded that there are few clear-cut general differences between littoral and sublittoral species in their behavioural and physiological adaptations to hypoxia; the main distinguishing feature of littoral-adapted species is their ability to control air-gaping. Changes in heart activity generally indicate variations in metabolic rate, the speed at which the metabolic rate may be altered reflecting the degree of adaptation to the littoral environment.     

Tissue culture of crownvetch (Coronilla varia L.) and the production of cardenolide-like substancesin vitro

The caryotype (2n = 24) of crownvetch (Coronilla varia L.) and the conditions of successful cultivation of tissue cultures derived from it were determined. The Murashige-Skoog cultivation medium with the addition of indol-3-ylacetic acid (1 mg 1¹), 1-naphthaleneacetic acid (0.1 mg 1¹), or coconut milk (20 %) was found to be the most suitable. A complex of cardenolide-like substances identical in its composition with that found in leaves of intact crownvetch plants was detected by means of thin layer chromatography in the extracts of these explant cultures up to the seventh subculture. These findings are also in agreement with the results of our earlier experiments in which strong pharmacological effects of cardenolides obtained from tissue cultures of this plant were demonstrated. Further investigations must be devoted to the optimalization of cultivation conditions of crownvetch cultures with the aim of their utilization as anin vitro source of cardioactive substances.     

Isolation and elemental analysis of metal-rich granules from the kidney of the scallop, Pecten maximus (L.)

Excretory granules from the kidney of the scallop, Pecten maximus (L.), have been isolated and purified. Their morphology has been determined by electron microscopy and their elemental composition by electron-probe X-ray micro-analysis. The granules, varying in size from 5–15 gmm diameter, are inorganic in nature and contain Ca, Mn, Zn, and P, equivalent to 75% of their dry weight, with Mg, Cu, Fe, Cd, K, S, and Cl also present in much smaller amounts. The granules show large variations in elemental composition but the sum of the metals is always close to equimolarity with the phosphorus content. Electron microscopy of the kidney tissue showed that there is a gradual development of the granules from lysosomal membranous vesicles to highly mineralized membrane-limited vacuoles or residual bodies, which are eventually excreted as such into the urinary tract.     

Incorporation of microalgae sterols by scallop Pecten maximus (L.) larvae

Changes in sterol composition of Pecten maximus larvae during the larval development stage with standard algal mixtures and unialgal diets were analysed. The sterol composition of four microalgae currently used in mollusc hatchery were also examined. Under standard algal conditions, the larvae quickly use the steryl ester from larvae reserves during the endotrophic and the mixotrophe phases. The preferential incorporation of Pavlova lutheri and T-Isochrysis sterols, rather than Skeletonema costatum sterols, during the larval development stage would indicate that S. costatum cells were poorly ingested and digested by larvae. Among the ingested sterols, cholesterol and stigmasterol were preferentially incorporated by the larvae. Conversely, the larvae appeared able to limit the incorporation of methylpavlovol, ethylpavlovol, and 4alpha-methylporiferasterol. In the unialgal experiment, the best growths were obtained with the diet richest in cholesterol (Chaetoceros calcitrans) and the best compromise of good growth and settlement rate was observed with the diet richest in C24 ethyl sterol. The selective incorporation of the cholesterol was confirmed by the larval rearing with C. calcitrans. The strong sterol dietary imprint in larvae corroborated the absence of an important capacity in P. maximus larvae to convert or biosynthesise sterol.     

Catecholaminergic responses to environmental stress in the hemolymph of Zhikong scallop Chlamys farreri

Catecholamines have long been known to play essential roles in several physiological processes in invertebrates. In this study, enzyme immunoassays were used to examine the levels of catecholamines (including adrenaline (AD), noradrenaline (NA) and dopamine (DA)) in the hemolymph of the scallop, Chlamys farreri, in responses to selected environmental stress. These stressors include high temperature (acute transfer from 17 to 28 degrees C), low salinity (acute transfer from salinity 31 to 20 psu) and air exposure, which are commonly encountered in aquaculture practices or in their natural habitat. The results showed that both AD and NA concentrations increased significantly in response to all the stressors. The levels of these catecholamines peaked 72 hr after acute temperature or salinity stresses, and then decreased gradually. For scallops subjected to air exposure, the duration of AD concentration elevation depended on the exposure temperature with a higher air temperature resulting in a shorter time to return to control level. In contrast, the NA responses were long lasting and no obvious decreases were observed after reaching the peak value. Interestingly, the DA concentration showed an inverse trend in response to stresses compared with AD or NA. These results suggested that the catecholaminergic responses in bivalve hemolymph will provide useful references in predicting the success of aquaculture practices and monitoring the environmental stresses.     

Development of twenty-two polymorphic microsatellite loci in the noble scallop, Chlamys nobilis

Twenty-two polymorphic microsatellites were isolated from the noble scallop, Chlamys nobilis with a partial genomic library enriched for tandem repeat sequences of (CA)₁₂ and (GA)₁₂. Polymorphism of these loci was assessed in a sample of 32 unrelated individuals. The average allele number was 7.23 per locus with a range of 3–19, and the observed and expected heterozygosity varied from 0.032–0.875 (averaging 0.452) and 0.315–0.939 (averaging 0.646), respectively. After Bonferroni correction (P > 0.0023), 11 of the 22 markers accorded with Hardy–Weinberg equilibrium, and the rest showed significant departure from HWE. Three pairs of loci showed significant linkage disequilibrium (P < 0.01). A poor transferability of these loci to other two cultured species, C. farreri and Patinopecten yessoensis was detected. These markers would be useful for studies of population genetics, linkage mapping and other relevant research in C. nobilis.     

Genetics of black gram (Phaseoulus mungo L.)

In black gram, the mode of inheritance of six pairs of characters has been analysed, viz.: erect versus spreading habit; hairy versus glabrous fruit; bluish black versus straw fruit colour; shiny versus dull seed surface; green versus brown seed colour. On the basis of the results, the following gene symbols are proposed:Erect habitSp is incompletely dominant over spreading,sp.—Hairy fruitG is dominant over glabrous,g, while inG plants the extent of hairiness is controlled by more than one pair of genes.—Bluish black fruit colourS is dominant over straw,s.—Shiny seed surfaceD is dominant over dull,d.—Green seed colourBr is dominant over brown;br.—Mosaic colour pattern of the seedUc is dominant over non-patterned,uc.Linkage was observed betweenS andUc, as well as betweenBr andD.     

Enzymatic proteinaceous hydrolyzate from the processing waste of the Iceland scallop Chlamys islandica]

Dry enzymatic hydrolyzate was derived from the protein containing wastes of the Iceland scallop fishery using the complex of proteinases from the king red crab hepatopancreas. The finished product contains not less than 80% of free amino acids and the lowest peptides. Leucine, isoleucine, arginine, lysine and aspartic acids account for to 50% of free amino acids. The applicability appraisal of proteinaceous hydrolyzate from the wastes of the Iceland scallop fishery to use them as the constituents in the nutrient medium for microorganisms was conducted. The approbation of hydrolyzate in the composition of nutrient medium on the test--cultures indicated the high sensibility and germinating capacity of microorganisms.     

Expressed sequence tags from the zhikong scallop (Chlamys farreri): Discovery and annotation of host-defense genes

A high-quality cDNA library was constructed from whole body tissues of the zhikong scallop, Chlamys farreri, challenged by Listonella anguillarum. A total of 5720 clones were sequenced, yielding 5123 expressed sequence tags (ESTs). Among the 3326 unique genes identified, 2289 (69%) genes had no significant (E-value < 1e?5) matches to known sequences in public databases and 194 (6%) matched proteins of unknown functions. The remaining 843 (25%) genes that exhibited homology with genes of known functions, showed broad involvement in metabolic processes (31%), cell structure and motility (20%), gene and protein expression (12%), cell signaling and cell communication (8%), cell division (4%), and notably, 25% of those genes were related to immune function. They included stress response genes, complement-like genes, proteinase and proteinase inhibitors, immune recognition receptors and immune effectors. The EST collection obtained in this study provides a useful resource for gene discovery and especially for the identification of host-defense genes and systems in scallops and other molluscs.