Investigation of molecular mechanisms regulating biomineralization of pearl oyster Pinctada maxima

Biomineralization is a process encompassing all mineral containing tissues produced within an organism. The most dynamic example of this process is the formation of the mollusk shell, comprising a variety of crystal phases and microstructures. The organic component incorporated within the shell is said to dictate this remarkable architecture. Subsequently, for the past decade considerable research have been undertaken to identify and characterize the protein components involved in biomineralization. Despite these efforts the general understanding of the process remains ambiguous. This study employs a novel molecular approach to further the elucidation of the shell biomineralization. A microarray platform has been custom generated (PmaxArray 1.0) from the pearl oyster Pinctada maxima. PmaxArray 1.0 consists of 4992 expressed sequence tags (ESTs) originating from the mantle, an organ involved in shell formation. This microarray has been used as the primary tool for three separate investigations in an effort to associate transcriptional gene expression from P. maxima to the process of shell biomineralization. The first investigation analyzes the spatial expression of ESTs throughout the mantle organ. The mantle was dissected into five discrete regions and each analyzed for gene expression with PmaxArray 1.0. Over 2000 ESTs were differentially expressed among the tissue sections, identifying five major expression regions. Three of these regions have been proposed to have shell formation functions belonging to nacre, prismatic calcite and periostracum. The spatial gene expression map was confirmed by in situ hybridization, localizing a subset of ESTs from each expression region to the same mantle area. Comparative sequence analysis of ESTs expressed in the proposed shell formation regions with the BLAST tool, revealed a number of the transcripts were novel while others showed significant sequence similarities to previously characterized shell formation genes. The second investigation correlates temporal EST expression during P. maxima larval ontogeny with transitions in shell mineralization during the same period. A timeline documenting the morphologicat microstructural and mineralogical shell characteristics of P. maxima throughout larval ontogeny has been established. Three different shell types were noted based on the physical characters and termed, prodissoconch I, prodissoconch 11 and dissoconch. PmaxArray 1.0 analyzed ESTs expression of animals throughout the larval development of P. maxima, noting up-regulation of 359 ESTs in association with the shell transitions from prodissoconch 1 to prodissoconch 11 to dissoconch. Comparative sequence analysis of these ESTs indicates a number of the transcripts are novel as well as showing significant sequence similarities between ESTs and known shell matrix associated genes and proteins. These ESTs are discussed in relation to the shell characters associated with their temporal expression. The third investigation uses PmaxArray 1.0 to analyze gene expression in the mantle tissue of P. maxima specimens exposed to sub-lethal concentrations of a shell-deforming toxin, tributyltin (TBT). The shell specific effects of TBT are used in this investigation to interpret differential expression of ESTs with respect to shell formation functions. A lethal and sublethal TBT concentration range was established for P. maxima, noting a concentration of 50 ng L- 1 TBT as sub-lethal over a 21 day period. Mantle tissue from P. maxima animals treated with 50 ng L- 1 TBT was assessed for differential EST expression with untreated control animals. A total of 102 ESTs were identified as differentially expressed in association with TBT exposure, comparative sequence identities included an up-regulation of immunity and detoxification related genes and down-regulation of several shell matrix genes. A number of transcripts encoding novel peptides were additionally identified. The potential actions of these genes are discussed with reference to TBT toxicity and shell biomineralization. This thesis has used a microarray platform to analyze gene expression in spatial, temporal and toxicity investigations, revealing the involvement of numerous gene transcripts in specific shell formation functions. Investigation of thousands of transcripts simultaneously has provided a holistic interpretation of the organic components regulating shell biomineralization.

Spatial analysis of biomineralization associated gene expression from the mantle organ of the pearl oyster Pinctada maxima

Background: Biomineralization is a process encompassing all mineral containing tissues produced within an organism. One of the most dynamic examples of this process is the formation of the mollusk shell, comprising a variety of crystal phases and microstructures. The organic component incorporated within the shell is said to dictate this architecture. However general understanding of how this process is achieved remains ambiguous. The mantle is a conserved organ involved in shell formation throughout molluscs. Specifically the mantle is thought to be responsible for secreting the protein component of the shell. This study employs molecular approaches to determine the spatial expression of genes within the mantle tissue to further the elucidation of the shell biomineralization. Results: A microarray platform was custom generated (PmaxArray 1.0) from the pearl oyster Pinctada maxima. PmaxArray 1.0 consists of 4992 expressed sequence tags (ESTs) originating from mantle tissue. This microarray was used to analyze the spatial expression of ESTs throughout the mantle organ. The mantle was dissected into five discrete regions and analyzed for differential gene expression with PmaxArray 1.0. Over 2000 ESTs were determined to be differentially expressed among the tissue sections, identifying five major expression regions. In situ hybridization validated and further localized the expression for a subset of these ESTs. Comparative sequence similarity analysis of these ESTs revealed a number of the transcripts were novel while others showed significant sequence similarities to previously characterized shell related genes.

Temporal tracking of mineralization and transcriptional developments of shell formation during the early life history of pearl oyster Pinctada maxima

Molluscan larval ontogeny is a highly conserved process comprising three principal developmental stages. A characteristic unique to each of these stages is shell design, termed prodissoconch I, prodissoconch II and dissoconch. These shells vary in morphology, mineralogy and microstructure. The discrete temporal transitions in shell biomineralization between these larval stages are utilized in this study to investigate transcriptional involvement in several distinct biomineralization events. Scanning electron microscopy and X-ray diffraction analysis of P. maxima larvae and juveniles collected throughout post-embryonic ontogenesis, document the mineralogy and microstructure of each shelled stage as well as establishing a timeline for transitions in biomineralization. P. maxima larval samples most representative of these biomineralization distinctions and transitions were analyzed for differential gene expression on the microarray platform PmaxArray 1.0. A number of transcripts are reported as differentially expressed in correlation to the mineralization events of P. maxima larval ontogeny. Some of those isolated are known shell matrix genes while others are novel; these are discussed in relation to potential shell formation roles. This interdisciplinary investigation has linked the shell developments of P. maxima larval ontogeny with corresponding gene expression profiles, furthering the elucidation of shell biomineralization.

Population structure and patterns of genetic variation in a pearl oyster (Pinctada radiata) native to the Arabian Gulf

The study assessed natural levels and patterns of genetic variation in Arabian Gulf populations of a native pearl oyster to define wild population structure considering potential intrinsic and extrinsic factors that could influence any wild structure detected. The study was also the first attempt to develop microsatellite markers and to generate a genome survey sequence (GSS) dataset for the target species using next generation sequencing technology. The partial genome dataset generated has potential biotechnological applications and for pearl oyster farming in the future.

History of the Atlantic Pearl-Oyster, Pinctata imbricata, Industry in Venezuela and Colombia, with Biological and Ecological Observations

In the 1500’s, the waters of Venezuela and to a lesser extent Colombia produced more natural pearls than any place ever produced in the world in any succeeding century. Atlantic pearl-oysters, Pinctata imbricata Röding 1798, were harvested almost entirely by divers. The pearls from them were exported to Spain and other European countries. By the end of the 1500’s, the pearl oysters had become much scarcer, and little harvesting took place during the 1600’s and 1700’s. Harvesting began to accelerate slowly in the mid 1800’s and has since continued but at a much lower rate than in the 1500’s. The harvesting methods have been hand collecting by divers until the early 1960’s, dredging from the 1500’s to the present, and hardhat diving from 1912 to the early 1960’s. Since the mid 1900’s, Japan and other countries of the western Pacific rim have inundated world markets with cultured pearls that are of better quality and are cheaper than natural pearls, and the marketing of natural pearls has nearly ended. The pearl oyster fishery in Colombia ended in the 1940’s, but it has continued in Venezuela with the fishermen selling the meats to support themselves; previously most meats had been discarded. A small quantity of pearls is now taken, and the fishery, which comprised about 3,000 fishermen in 1947, comprised about 300 in 2002.

A History of the Pearl Oyster Fishery in the Archipielago de las Perlas, Panama

The pearl oyster, Pinctada margaritifera mazatlanica, was once found around the Archipielago de las Perlas in Panama in abundance and it supported a substantial fishery by hard hat divers. The products were pearls, shells used for making buttons, and meats used locally for food. After the mid 1920’s, the fishery declined due to overfishing, and by the 1940’s it was nearly gone. The oysters began to repopulate the grounds during the 1970’s, but the oysters remain relatively scarce. Fishing has since resumed on a small scale by skin divers using face masks.

Pearl culture and pearl oyster cultivation

Few marine animals have yet been tamed and harnessed for man's use. Of these the bivalves have the greatest potential, and of the bivalves the pearl oysters are perhaps the most thoroughly exploited. Not only are they eaten and their shell put to use, but their nacre secreting properties are tapped to produce pearls by design, rather than by accident as in nature.

Pearl oyster survey, Gulf of Mannar, 1964

This note reports the results of the 1964 Pearl Bank Survey. These surveys are conducted annually by the Fisheries Department to assess the possibility of a fishery; for detailed accounts of past surveys see Sivalingam (1955, 1958) who has also provided (1962) a complete list of all literature on the Ceylon Pearl Banks. The 1964 survey was carried out with the Department's m. v. "North Star" (42') and m. v. "Seer" (45') and a 25' mechanised fishing boat as tender, between the 17th and 29th of April.

The 1958 pearl oyster fishery, Gulf of Mannar

The continental shelf between the three and twelve fathom lines, off the Ceylon coast in the Gulf of Mannar is popularly known as the Pearl Banks. Though the area of this plateau is extensive the pearl oysters are concentrated on restricted areas known as "paars". The more productive of these are the Cheval Group of paars and the Moderagam Paar. The Peria Paar and Twynam's Paar though covering larger areas are not dependable as oyster beds. The True Vankalai, Peria Paar Karai and Muthuvarathu Paars are small in extent, but have yielded oysters on a commercial scale. Although the Ceylon Pearl Banks has been commercially fished for centuries, the first scientific investigation of its pearl oysters on an intensive scale was started by Herdman in 1902 (1902-06) and later continued by Hornell (1905-41), Shipley (1904-06), Pearson (1911-33), Jameson (1912-13), Southwell (1910-14) and Malpas (1922-39).

Effects of hydrocarbons on reproductive physiology of the pearl oyster, Pinctada fucata

There are a lot of evidence that show hvdrocarbones cause some defect in reproduction and growth of bivalves. Bivalves are filter-feeder, thus accumulate more hydrocarbones in their tissue. In this study adult pearl producing oysters (Pinctada fucata) are used for all experimens. Samples of oysters, water and sediment from four natural beds; Nakhiloo (clean), Hendurabi (semipolluted), Lavan 1 (semipolluted) and Lavan 2 (polluted) were gatherd for 13 succesive months. Temperature, salinity, pH, oxygen and turbidity were recorded in each sampling. Oysters were kept in laboratory for adapation and then their length (DVM) were measured. Hemolymph samples were collected by insuline syring. Sediments and soft tissues of oysters were dissolved in carbon tetrachloride and when heated to extract oil hydrocarbones. UV, GC and IR were used to assay oil hydrocarbones. Accumulation of hydrocabones in soft tissue were as follows : Kakhiloooysters, major one in late spring and minor one in mid fall. Little elevation of progesterone start gamete and a lot of elevation release gametes_ Esteradiols gradually increase during gametogenesis and reach to maximum level during vitellogenesis. Testosterone have a synergestic role which esteradiol during vitellogenesis and also is effective in male sexuality. Multi regression test showed that there isn't meaning relationship (P<0.05) between hydrocarbones pollution and esteradiols. However in Lavan 2 due to hormone concentration of hydrocarbones had some effect in sexual hormonal cycles. Turbidity was the most effective factor for releasing of progesterone. Progesterone was a stimulating factor for releasing estradiol also release of testosterone was corrolated with oxygen and depth of beds. One way analysis of variance showed that there is not significant relasionship (P<0.05) between different factory in stations.

Integrated cultivation of the red alga Kappaphycus alvarezii and the pearl oyster Pinctada martensi

The effect of simultaneously cultivating the pearl oyster Pinctada martensi and the red alga Kappaphycus alvarezii on growth rates of both species was investigated in laboratory and field studies conducted from December 1993 to June 1995. The two study sites were in subtidal areas 100 km apart off the east coast of Hainan Island, China. Pearl oysters were cultivated in the center of an algal farm and red alga was cultivated in the center of the pearl oyster farm. These field experiments showed higher growth rates of both P. martensi and K. alvarezii in a co-culture system than in a monospecies culture system. Laboratory studies showed that the algae removed nitrogenous wastes released by pearl oysters. Algae treated with pearl oyster wastes grew much faster than those without oyster wastes. Algae treated with the seawater to which NH4Cl, NaNO3 and NaNO2 were added grew at the same rate as those treated with natural seawater containing oyster nitrogenous wastes, suggesting that enhanced growth of algae in the co-culture system was largely due to nitrogenous metabolites of the pearl oysters. In the co-culture, growth of pearl oysters was positively influenced by the presence of rapidly growing algae but when seawater temperature decreased below 20 degrees C, the algae grew slowly and there was no measurable benefit of mixed culture to either algae or pearl oyster.

Studies on The Eco Biology of The Indian Pearl Oyster, Pinctada Fucata (Gould)

The present work aims to study induced maturation of the pearl oyster for induced spawning experiments. The work on larval development was done with a view to developing techniques for the artificial rearing of commercially important pearl oyster P fucata, and also to elucidate the principles and problems of tropical bivalve larvae in general for detailed investigations in the future. The present study is designed to probe into the details of the basic aspects of the biology related to the hatchery technology of Pinctada fucata and the understanding of the factors which influence induction of maturation, spawning, larval rearing and spat settlement. This would go a long way in the upgradation of hatchery technology of the Indian Pearl oyster Pinctada fucata fora commercial level seed production..

Studies on Larval Nutrition in The Pearl Oyster Pinctada Pucata (Gould)

The present study has yielded a great deal of information on nutrition of pearl oyster larvae. T he formulae presented may be used effectively and with advantage in improving the larval rearing system with specific reference to nutritional aspects. It is also hoped that this is the first comprehensive study on pearl oyster larval nutrition would stimulate further detailed investigations on many of the other finer aspects of tropical bivalve larval nutrition.

Molecular genetic profiling of functional genes of pearl oyster, Pinctada fucata

There are a number of genes involved in the regulation of functional process in marine bivalves. In the case of pearl oyster, some of these genes have major role in the immune/defence function and biomineralization process involved in the pearl formation in them. As secondary filter feeders, pearl oysters are exposed to various kinds of stressors like bacteria, viruses, pesticides, industrial wastes, toxic metals and petroleum derivatives, making susceptible to diseases. Environmental changes and ambient stress also affect non-specific immunity, making the organisms vulnerable to infections. These stressors can trigger various cellular responses in the animals in their efforts to counteract the ill effects of the stress on them. These include the expression of defence related genes which encode factors such as antioxidant genes, pattern recognition receptor proteins etc. One of the strategies to combat these problems is to get insight into the disease resistance genes, and use them for disease control and health management. Similarly, although it is known that formation of pearl in molluscs is mediated by specialized proteins which are in turn regulated by specific genes encoding them, there is a paucity of sufficient information on these genes.In view of the above facts, studies on the defence related and pearl forming genes of the pearl oyster assumes importance from the point of view of both sustainable fishery management and aquaculture. At present, there is total lack of sufficient knowledge on the functional genes and their expressions in the Indian pearl oyster Pinctada fucata. Hence this work was taken up to identify and characterize the defence related and pearl forming genes, and study their expression through molecular means, in the Indian pearl oyster Pinctada fucata which are economically important for aquaculture at the southeast coast of India. The present study has successfully carried out the molecular identification, characterization and expression analysis of defence related antioxidant enzyme genes and pattern recognition proteins genes which play vital role in the defence against biotic and abiotic stressors. Antioxidant enzyme genes viz., Cu/Zn superoxide dismutase (Cu/Zn SOD), glutathione peroxidise (GPX) and glutathione-S-transferase (GST) were studied. Concerted approaches using the various molecular tools like polymerase chain reaction (PCR), random amplification of cDNA ends (RACE), molecular cloning and sequencing have resulted in the identification and characterization of full length sequences (924 bp) of the Cu/Zn SOD, most important antioxidant enzyme gene. BLAST search in NCBI confirmed the identity of the gene as Cu/Zn SOD. The presence of the characteristic amino acid sequences such as copper/zinc binding residues, family signature sequences and signal peptides were found out. Multiple sequence alignment comparison and phylogenetic analysis of the nucleotide and amino acid sequences using bioinformatics tools like BioEdit,MEGA etc revealed that the sequences were found to contain regions of diversity as well as homogeneity. Close evolutionary relationship between P. fucata and other aquatic invertebrates was revealed from the phylogenetic tree constructed using SOD amino acid sequence of P. fucata and other invertebrates as well as vertebrates