Soluble organic matrices of the calcitic prismatic shell layers of two Pteriomorphid bivalves. Pinna nobilis and Pinctada margaritifera

The calcitic prisms of the shells of two bivalves, Pinna and Pinctada, are considered simple prisms according to some morphological and mineralogical characteristics. Scanning electron microscopic and atomic force microscopic studies show that the microstructures and nanostructures of these two shells are different. Pinna prisms are monocrystalline, whereas Pinctada prisms are not. Moreover, intraprismatic membranes are present only in the Pinctada prisms. The soluble organic matrices extracted from these prisms are acidic, but their bulk compositions differ. Ultraviolet and infrared spectrometries, fluorescence, high pressure liquid chromatography, and electrophoresis show that the sugar-protein ratios and the molecular weights are different. Sulfur is mainly associated with acidic sulfated sugars, not with amino acids, and the role of acidic sulfated sugars is still underestimated. Thus, the simple prism concept is not a relevant model for the biomineralization processes in the calcitic prismatic layer of mollusk shells.     

江珧活性蛋白的分离及抗氧化作用研究

采用匀浆、硫酸铵分级沉淀和Sephadex G-100柱层析的方法从江珧的闭壳肌及内脏团分别分离到3个和2个活性蛋白峰,经化学发光法进行体外抗氧化测定,这些活性蛋白都具有清除超氧阴离子自由基、羟自由基、过氧化氢和抑制脂质过氧化作用,其中闭壳肌第三个蛋白峰(PB3)与内脏团第二个蛋白峰(PN2)抑制能力最强。     

Development of 26 highly polymorphic microsatellite markers for the highly endangered fan mussel Pinna nobilis and cross-species amplification

Molecular Biology Reports - The fan mussel, Pinna nobilis is a highly endangered bivalve species endemic to the Mediterranean Sea. During the last few decades, populations have been greatly reduced...     

Anaerobiosis and metabolic plasticity of Pinna nobilis: Biochemical and ecological features

Changes in the energetic metabolism were studied in the fan mussel Pinna nobilis L. exposed to environmental and anthropic stress. The high polymorphism of enzymes suggests an adaptation of the fan mussel to environmental variability peculiar of transitional waters with respect to the same species living in exposed coastal sea. The electrophoretic patterns showed a predominance of LDH-A4 and the presence of both mitochondrial and cytosolic MDH isozymes. Moreover, in all the analyzed tissues and organs, MDH activity was greater than the LDH one. Metabolic plasticity of the fan mussel is further highlighted by octopine dehydrogenase and superoxide dismutase electrophoretic patterns, showing the presence of many isoforms. These evidences are also confirmed by spectroscopic determinations of alanopine, tauropine, strombine and octopine dehydrogenase activity characterized by a specific trend due to environmental variability. Specific variations in anaerobic capacity of P. nobilis L. are discussed in relation to their distribution according to the marine-brackish gradient.     

Genetic variation among populations of the endangered fan mussel <Emphasis Type="Italic">Pinna nobilis</Emphasis> (Mollusca: Bivalvia) along the Tunisian coastline

The Mediterranean fan mussel Pinna nobilis is an endangered invertebrate due to overexploitation and habitat deterioration. In this species, two distinctive morphs, the “combed” and “straight and wide” forms, have been recorded and it is not known whether this morphometric variability is attributed to genetic or environmental factors. In this study, we used mitochondrial COI sequences to describe the genetic variability of five Pinna nobilis populations sampled along the northern, eastern, and southern Tunisian coastline, and to examine whether there is a genetic differentiation between the two forms of this species. DNA sequences of 675 bp from the mitochondrial COI gene have revealed 10 different haplotypes among 49 examined specimens. Haplotype diversity was high, ranging between 0.40441 and 0.80952, and showed a decreasing North-East gradient, which seems to be explained by the hydrogeography of the study area. The mitochondrial COI marker did not suggest a genetic distinctiveness between the two Pinna nobilis shell forms, “combed” and “straight and wide”. Although the morphometric plasticity of the fan mussel could be due to the influence of environmental factors, further genetic studies using nuclear markers are envisaged to investigate whether this differentiation is associated to gene flow restrictions.     

Comparison of the soluble matrices of the calcitic prismatic layer of Pinna nobilis (Mollusca,Bivalvia, Pteriomorpha)

The calcitic prisms of the outer layer of the shell of Pinna nobilis, surrounded by thick organic walls, contain a soluble intracrystalline matrix. The structure and composition of the outer interprismatic walls are not well known. The current viewpoint is they are composed of an insoluble matrix. Another thick organic structure, the interlamellar sheet of the nacreous layer, is composed of insoluble and soluble matrices. The composition of two sets of soluble organic matrices from the calcitic layer of Pinna nobilis, extracted with and without the organic walls are compared. According to the various analyses (SEM and AFM, UV and FTIR spectrometry, HPLC, electrophoreses, XANES), the main characteristics of the two matrices are similar, but not identical. Thus, the organic walls contain soluble components. However, the three-layered structure of the interlamellar sheet of the nacreous layer has not been observed.     

Mitochondrial DNA Reveals Genetic Structuring of Pinna nobilis across the Mediterranean Sea

Pinna nobilis is the largest endemic Mediterranean marine bivalve. During past centuries, various human activities have promoted the regression of its populations. As a consequence of stringent standards of protection, demographic expansions are currently reported in many sites. The aim of this study was to provide the first large broad-scale insight into the genetic variability of P. nobilis in the area that encompasses the western Mediterranean, Ionian Sea, and Adriatic Sea marine ecoregions. To accomplish this objective twenty-five populations from this area were surveyed using two mitochondrial DNA markers (COI and 16S). Our dataset was then merged with those obtained in other studies for the Aegean and Tunisian populations (eastern Mediterranean), and statistical analyses (Bayesian model-based clustering, median-joining network, AMOVA, mismatch distribution, Tajima’s and Fu’s neutrality tests and Bayesian skyline plots) were performed. The results revealed genetic divergence among three distinguishable areas: (1) western Mediterranean and Ionian Sea; (2) Adriatic Sea; and (3) Aegean Sea and Tunisian coastal areas. From a conservational point of view, populations from the three genetically divergent groups found may be considered as different management units.     

Caspartin and calprismin, two proteins of the shell calcitic prisms of the Mediterranean fan mussel Pinna nobilis

We used the combination of preparative electrophoresis and immunological detection to isolate two new proteins from the shell calcitic prisms of Pinna nobilis, the Mediterranean fan mussel. The amino acid composition of these proteins was determined. Both proteins are soluble, intracrystalline, and acidic. The 38-kDa protein is glycosylated; the 17-kDa one is not. Ala, Asx, Thr, and Pro represent the dominant residues of the 38-kDa protein, named calprismin. An N-terminal sequence was obtained from calprismin. This sequence, which comprises a pattern of 4 cysteine residues, is not related to any known protein. The second protein, named caspartin, exhibits an unusual amino acid composition, since Asx constitutes by far the main amino acid residue. Preliminary sequencing surprisingly suggests that the first 75 N-terminal residues are all Asp. Caspartin self-aggregates spontaneously into multimers. In vitro tests show that it inhibits the precipitation of calcium carbonate. Furthermore, it strongly interferes with the growth of calcite crystals. A polyclonal antiserum raised against caspartin was used to localize this protein in the shell by immunogold. The immunolocalization demonstrates that caspartin is distributed within the prisms and makes a continuous film at the interface between the prisms and the surrounding insoluble sheets. Our finding emphasizes the prominent role of aspartic acid-rich proteins for the building of calcitic prisms among molluscs.     

Abundance and growth of Pinna carnea (Mytiloida: Pinnacea) juveniles in suspended-frame culture

We quantified the abundance of juvenile Pinna carnea at Turpialito, Golfo de Cariaco, Venezuela, using collectors suspended at 8 m depth for 16 months and then evaluated their subsequent growth in suspended enclosures over 14 months in relation to concurrent measures of temperature and planktontonic food availability. All body components showed a rapid increase during the first four-five months. There was a decrease in somatic tissue mass and great variability in growth coincident at first spawning, suggesting a negative effect of gonadal production on somatic growth. A multiple regression analysis indicated a strong relationship between tissue growth and food availability as measured by organic sestonic matter. Although we determinated an adequated spat abundance for culture activities (80 spat per collector-30 x 60 cm), spat collection was limited, mainly from February through April. In addition to high variability in growth and negative influence of environmental factors during unfavorable periods, the muscles production required in 14 months, suggesting that P. carnea is not appropriate for commercial production of muscle. However, the high weight of all edible tissues mass attained (21 g) and the high survival rate in all experimentation (> 96%) suggests that P. carnea is fit for aquaculture.     

Antioxidant response of the bivalve Pinna nobilis colonised by invasive red macroalgae Lophocladia lallemandii

Invasive species represent a risk to natural ecosystems and a biodiversity hazard. The present work aims to determine the antioxidant enzyme response – superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX), the phase II detoxifying enzyme – glutathione S-transferase (GST) – and markers of oxidative damage – thioredoxin reductase (TR) and malondialdehyde (MDA) – in gills and digestive gland of Pinna nobilis and to study the antioxidant response effects in the bivalve colonised by the invasive macroalgae Lophocladia lallemandii. Colonised specimens were collected in a control area without L. lallemandii and another area completely colonised by L. lallemandii. All enzyme activities were found to be present in gills and digestive gland, with some tissue differences. CAT and SOD activities were higher in gills than digestive gland, whereas GST activity and MDA levels were higher in digestive gland. The presence of L. lallemandii induced a significant increase in the activities of antioxidant enzymes in both gills and digestive gland, except for CAT activity in gills. GST and TR activities were also increased in both tissues, as well as the MDA concentration. We can conclude that the presence of L. lallemandii colonising P. nobilis induces a biological stress and oxidative damage to the fan mussel.     

In situ chemical speciation of sulfur in calcitic biominerals and the simple prism concept

The microstructure and composition of two mollusc shells were investigated using a combination of light microscopy, SEM, EPMA, and XANES. The shells of Pinna and Pinctada are composed of calcite prisms separated by organic walls. The prismatic units of Pinna are monocrystalline, and those of Pinctada are polycrystalline with internal organic radial membranes. High-spatial-resolution XANES maps for the different S species across adjacent prisms show that sulfate is the principal component in both the intraprismatic organic matrices and the outer membranes. Additionally, these maps confirm that the inner structures of the prismatic units are different for both genera. In many ways, the prisms of Pinna and Pinctada are different and invalidate the "simple prism" concept.     

On the biology of the Watchman prawn, Anchistus custos (Crustacea; Decapoda; Palaemonidae), an Indo-West Pacific commensal of the bivale Pinna

Anchistus custos Forskål is the only common prawn commensal with bivalves of the family Pinnidae in Singapore waters. The whole morphology shows pronounced modifications connected with the commensal mode of life. The second legs are large and massive and show marked positive allometry in the male but appear to have little function in the general life of the animal. The animal occurs wherever there are established populations of matureindividuals of its host. Infestation rates are heavy and are affected by size of host and probably by competition with pinnotherid crabs. Multiple infestations are common and always involve individuals of both sexes and it has not been possible to demonstrate aggressive or territorial behaviour. Reproduction appears to occur throughout the year. Individuals are not normally found away from their hosts but rapidly abandon dead hosts. Location of the host depends in part on tactile stimuli and thigmotaxy and in part on pronounced positive rheotaxy. It has not been possible to demonstrate any distance chemical sense involved in host location but the prawn is repulsed by the presence of dead Pinna flesh. The prawn normally clings to the edge of the gill lamellae of its host and feeds by means of the highly modified first legs which are used as scrapers to remove mucus and entangled food particles from the gills of the host. Examination of gut contents of the prawn confirms this deduction. There is no evidence for any harmful effects on the host.     

Occupation of patches in the epifaunal communities on pier pilings and the bivalve Pinna bicolor at Edithburgh,South Australia

Summary The reoccupation of artificially cleared patches in a subtidal epifaunal community was investigated in two field experiments on the pilings of Edithburgh pier, South Austrlia. In most cases, the greatest proportion of the patch was reoccupied by the vegetative extension of established sponge and tunicate colonies adjacent to it. Larval recruitment by sponges, bryozoans, tunicates and serpulids contributed to the reoccupation but resulted in only a small proportion of the mean percentage cover. The relative abundances of individual species established in any patch were shown to be a function of the (1) position in space, (2) age, (3) time of creation, (4) initial size of the patch.There was a large amount of between-patch variation in all cases. Overgrowth interactions occurred frequently within patches, and for many pairs of species, neither species consistently overgrew the other. Overgrowth interactions were tested statistically, and a large number of pairs of species were found to be competitively equivalent. This represents a possible situation additional to the alternatives recognized in the literature, namely competitive hierarchies or networks. Interactions between species should be regarded as stochastic, with a wide range of possible outcomes. The situation at Edithburgh is likely to produce greater between-patch variability than either a network or a hierarchy.Despite this large variation, super-specific taxa differ fairly consistently in capacity for overgrowth. Tunicates overgrow sponges, which overgrow bryozoans, which overgrow serpulids. The occupation of most patches was directional in the sense that bryozoans and serpulids invaded first, but tunicates and sponges excluded them and came to dominate the patch. These realtionships are used to predict patterns of abundance for substrata which are small and isolated, and these predictions are compared with the epifauna of the bivalve Pinna bicolor, which provides such substrata adjacent to the pier.     

Point pattern analysis as a tool for assessing disease spread and population features in remaining sanctuaries of the critically endangered bivalve Pinna nobilis

An emergent disease has relegated populations of the Mediterranean pen shell, Pinna nobilis L. critically endangered to sanctuaries featuring salinities outside the 36.5 to 39 range. Point pattern analysis was used in three areas of the Alfacs Bay (Ebro Delta) still hosting pen shells to assess the possible undergoing of disease spread by comparing the spatial distribution of live individuals vs. empty shells across spatial scales. We also evaluated the importance of other ecological aspects of conservation relevance such as the size distribution of individuals, and the possible association to seagrass habitats. The population assessment showed no recent mortality and a clear dominance of large adults among empty shells (97.3%) pointing to no disease spread during the study period. At the low spatial scale Nearest Neighbor (NN) analyses evidenced significant clustering (NN Ratios of 0.4–0.8), but in one of the zones NN distances were closer in empty shells than in live individuals, suggesting a former localized outbreak. At the larger spatial scale, MDSCA confirmed clustering patterns up to distances of 115 to 190 m, with higher aggregation of empty shells at the same study zone. The bay also featured low juvenile availability (3.2%), which risks the continuity of the population. No evidence for habitat or conspecific selection could be observed from abundance patterns and variation in NN across study regions. Our research provides a tool for assessing population condition in paralic environments, where salinity conditions tend to slow down disease spread, thus allowing a time gap for undertaking conservation decisions.     

Genetic structure of the endangered species <Emphasis Type="Italic">Pinna nobilis</Emphasis> (Mollusca: Bivalvia) inferred from mtDNA sequences

This study examines the population genetic structure of the endangered bivalve Pinna nobilis (Mollusca: Bivalvia), based on novel mtDNA sequences (partial COI and 16S rDNA mtDNA genes). The analyzed nucleotide sequences of COI were 729 bp in size, coding for a 243 amino acid peptide, while the analyzed nucleotide sequences of 16S rDNA were 489 bp in size. These sequences of P. nobilis were the first DNA sequences of the species submitted to any Genetic Data Base. Population samples from four geographic regions from Greece, as well as a population sample of Atrina fragilis (as an outgroup) were used. High values of haplotypic diversity were found in the population samples of P. nobilis, based on the COI sequences. A single base in the analyzed 16S rDNA sequences was different in all analyzed individuals from a single population sample (Chios island) differentiating it from the other ones. These mtDNA sequences could be informative for further genetic analyses of the endangered species, contributing in conservation plans for its protection and/or aquaculture investigations.     

托叶铁科(苏铁目)羽片脉序和羽片解剖学研究

详细研究了托叶铁科两属的2种代表植物Stangeria eriopus和Bowenia serrulata的羽片脉序和羽片解剖学,两个属在气孔器特征与不具副传输组织方面极为相似。而在气孔的分布,羽片脉序式样与叶缘形态,粘液道的有无,海绵组织与栅栏组织的分化。工字厚壁组织与表皮细胞垂周壁特征方面则有较大的差异。将这些特征与苏铁科和泽米铁科作了比较,并讨论了这些特征在系统学上的意义。研究结果支持Stevenson(1992)将该科分为两亚科以及托叶铁科是介入苏铁科与泽米铁科之间的观点。     

Studien über pinnaperlen.

Zusammenfassung Unter den von Dr. F. Weser in Holländisch-Indien auf der Insel Buton erworbenen, eigentümlich gefärbten Perlen (vgl. I. Mitteilung, Tafel I, Abb. 5–9) fand sich ein Stück, das nach Struktur und Optik durchaus mit Prismenperlen von Pinna nobilis übereinstimmte, dagegen in der am Schliff weinroten, nur dem Kalk (nicht dem Conchin) zukommenden Färbung sich von den Perlen der rotschaligen Steckmuscheln (z. B. Pinna nobilis) auffallend unterscheidet. Eine solche Färbung ist a ber der Prismenschicht von Pinna nigrina eigentümlich und daraus wird geschlossen, daß die untersuchte Perle der schwarzen Steckmuschel zugehört. Zugleich ergab sich damit die Möglichkeit, die entsprechend gefärbten Perlmutterperlen in dem genannten Material als solche von Pinna nigrina zu identifizieren. Weiter wurden Fälschungen solcher Perlen beschrieben, die in Wirklichkeit aus der Prismenschicht von Pinna nigrina künstlich hergestellte Kügelchen sind. Diese Imitationen, deren Wesen aus dem Schliffbild in gewöhnlichem und polarisiertem Lichte ohne weiteres verständlich wird, lassen sich bereits bei Untersuchung der Stücke in toto erkennen, indem bei genügend starker durchfallender Beleuchtung die von einer echten Perle abweichende parallelschichtige farbige Bänderung, die ohne Beziehung zur Kugelgestalt ist, wahrgenommen werden kann.     

泽米科植物羽片脉序和解剖学及其系统学意义

研究了苏铁目泽米科Zamiaceae 2亚科的所有4族(Stevenson系统, 1992)共10种代表植物的羽片脉序及解剖学特征,结果显示泽米科羽片脉序为二歧分叉的平行脉,无中脉。小刺双子铁Dioon spinulosum、大头非洲铁Encephalartos friderici-guilielmii和摩尔大泽米Macrozamia moorei等的平行脉末端以不同的形式互相连接,而鳞木铁Lepidozamia peroffskyana、粗壮角果铁Ceratozamia mexicana var. robusta、竹叶角果铁C. hildae、佛州泽米Zamia　floridana、柔叶泽米Z. debilis、鳞秕泽米Z. furfuracea和短尖泽米Z. muricata等的平行脉末端不连接而直达叶缘,其中鳞木铁、粗壮角果铁和竹叶角果铁的脉达叶缘后逐渐消失。羽片的横切面结构通常由表皮、下皮厚壁细胞和叶肉组成,表皮层包括上、下表皮各一层,叶肉可能同时分化出近上表面的栅栏组织和近下表面的栅栏组织,或仅有近上表面的栅栏组织分化,或无栅栏组织分化而完全为海绵组织。然而,泽米科没有典型的海绵组织和传输组织分化。小刺双子铁、大头非洲铁、鳞叶木铁和摩尔大泽米的羽片具有粘液道而无工字厚壁组织,在小刺双子铁中粘液道与维管束对生,在另3种中则与维管束轮生;但粗壮角果铁、竹叶角果铁、佛州泽米、柔叶泽米、鳞秕泽米和短尖泽米的羽片则具有工字厚壁组织而没有粘液道,其中粗壮角果铁和竹叶角果铁的羽片工字厚壁组织仅与上表皮相连,而佛州泽米、柔叶泽米、鳞秕泽米和短尖泽米的羽片工字厚壁组织与上、下表皮都相连。羽片脉序和解剖学特征支持Stevenson将泽米铁科分为两亚科的观点。     

Mucins and molluscan calcification. Molecular characterization of mucoperlin, a novel mucin-like protein from the nacreous shell layer of the fan mussel Pinna nobilis (Bivalvia, pteriomorphia)

A cDNA expression library constructed from mantle tissue mRNA of the Mediterranean fan mussel Pinna nobilis was screened with antibodies raised against the acetic acid-soluble shell matrix of the same species. This resulted in the isolation of a 2138-base pair cDNA, containing 13 tandem repeats of 93 base pairs. The deduced protein has a molecular mass of 66.7 kDa and a isoelectric point of 4.8. This protein, which is enriched in serine and proline residues, was overexpressed, purified, and used for producing polyclonal antibodies. Immunological in situ and in vitro tests showed that the protein is localized in the nacreous aragonitic layer of P. nobilis, but not in the calcitic prisms. Because this protein of the nacre of P. nobilis exhibits some mucin-like characteristics, we propose the name mucoperlin. This is the first paper reporting the cloning of a molluscan mucin and the first molecular evidence for the involvement of a mucin in molluscan calcification. This finding corroborates our previous hypothesis that some of the proteinaceous constituents of the molluscan shell matrix would derive from mucins, common to many metazoan lineages of the late Precambrian (Marin, F., Smith, M., Isa, Y., Muyzer, G. and Westbroek, P. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 1554-1559). The adaptation of an ancestral mucin to a new function, the regulation of the mineralization process, may be one of the molecular events, among others, that would explain the simultaneous emergence of organized calcification in many metazoan lineages during the Cambrian explosion.