Mollusca evolutionary success can be attributed partly to their efficiency to sustain and protect their soft body with an external biomineralized structure, the shell. Current knowledge of the protein set responsible for the formation of the shell microstructural polymorphism and unique properties remains largely patchy. In Pinctada margaritifera and Pinctada maxima, we identified 80 shell matrix proteins, among which 66 are entirely unique. This is the only description of the whole "biomineralization toolkit" of the matrices that, at least in part, is thought to regulate the formation of the prismatic and nacreous shell layers in the pearl oysters. We unambiguously demonstrate that prisms and nacre are assembled from very different protein repertoires. This suggests that these layers do not derive from each other.mantle | mollusk shell matrix proteins | proteome | transcriptome | evolution

BackgroundThe shell of the pearl-producing bivalve Pinctada margaritifera is composed of an organic cell-free matrix that plays a key role in the dynamic process of biologically-controlled biomineralization. In order to increase genomic resources and identify shell matrix proteins implicated in biomineralization in P. margaritifera, high-throughput Expressed Sequence Tag (EST) pyrosequencing was undertaken on the calcifying mantle, combined with a proteomic analysis of the shell.ResultsWe report the functional analysis of 276 738 sequences, leading to the constitution of an unprecedented catalog of 82 P. margaritifera biomineralization-related mantle protein sequences. Components of the current "chitin-silk fibroin gel-acidic macromolecule" model of biomineralization processes were found, in particular a homolog of a biomineralization protein (Pif-177) recently discovered in P. fucata. Among these sequences, we could show the localization of two other biomineralization protein transcripts, pmarg-aspein and pmarg-pearlin, in two distinct areas of the outer mantle epithelium, suggesting their implication in calcite and aragonite formation. Finally, by combining the EST approach with a proteomic mass spectrometry analysis of proteins isolated from the P. margaritifera shell organic matrix, we demonstrated the presence of 30 sequences containing almost all of the shell proteins that have been previously described from shell matrix protein analyses of the Pinctada genus. The integration of these two methods allowed the global composition of biomineralizing tissue and calcified structures to be examined in tandem for the first time.ConclusionsThis EST study made on the calcifying tissue of P. margaritifera is the first description of pyrosequencing on a pearl-producing bivalve species. Our results provide direct evidence that our EST data set covers most of the diversity of the matrix protein of P. margaritifera shell, but also that the mantle transcripts encode proteins present in P. margaritifera shell, hence demonstrating their implication in shell formation. Combining transcriptomic and proteomic approaches is therefore a powerful way to identify proteins involved in biomineralization. Data generated in this study supply the most comprehensive list of biomineralization-related sequences presently available among protostomian species, and represent a major breakthrough in the field of molluskan biomineralization.

Bonamia sp. is a pathogenic parasite that occurs in the haemocytes of dredge oysters Ostrea chilensis Philippi in New Zealand. Ultrastructurally it resembles other haplosporidians in the possession of haplosporosomes, haplosporogenesis, persistence of mitotic microtubules during interphase and of the nuclear envelope during mitosis, and occurrence of a diplokaryotic or multi-nucleate plasmodial stage. Another stage containing a large vacuole derived from enlargement of 1 or more mitochondria has not previously been described from other haplosporidians. It most closely resembles B. ostreae Pichot et al., 1979, which parasitises and is pathogenic in haemocytes of European flat oysters, O. edulis. However, B. ostreae is smaller and denser, and has fewer lipoid bodies and haplosporosomes. We have nearly completely sequenced the small ribosomal gene of the organism from O. chilensis. Initial comparisons of these sequences with those of other protozoans showed similarities to B. ostreae. Polymorphism within Bonamia sp. was confirmed by restriction fragment length polymorphism analysis. On the basis of ultrastructural and molecular considerations it is proposed that this organism be named Bonamia exitiosus sp. nov. KEY WORDS: Bonamia exitiosus · Ostrea chilensis · Ultrastructure · 18S rDNA · New Zealand Resale or republication not permitted without written consent of the publisherDis Aquat Org 47: [63][64][65][66][67][68][69][70][71][72] 2001 MATERIALS AND METHODS Ultrastructural studies. Infected Ostrea chilensis (n = 237) from Foveaux Strait were opened one at a time, the heart was removed and cut in two, imprints were made with one half, and the other half was fixed for ultrastructural studies. Imprints were stained with Hemacolor™ (Merck) and microscopically examined, and heavily infected oysters were selected for further study. Hearts were fixed in 2.5% glutaraldehyde in 0.22 µm filtered seawater for 1 to 2 h, washed twice in filtered seawater, post-fixed in 1% OsO 4 for 1 h, stained en bloc with 5% uranyl acetate in 0.1 M sodium acetate buffer for 45 min, dehydrated in ascending (50 to 100%) ethyl alcohol, embedded in Araldite, sectioned, stained with 5% uranyl acetate for 10 min and in 5% lead citrate for 5 to 6 min, and examined on a Philips 420ST TEM.To compare Bonamia sp. with B. ostreae, quantitative data were taken from published studies on the ultrastructure of B. ostreae (Pichot et al. 1979, Brehélin et al. 1982, Balouet et al. 1983, Grizel 1985, Hervio et al. 1991 and from archived material held at the IFREMER laboratory at Ronce-les-Bains, La Tremblade, France. Samples were compared directly under the TEM from blocks of B. ostreae infected Ostrea edulis kindly lent by Steve Feist of the MAFF Fish Diseases Laboratory, Weymouth, England.Base sequence studies. DNA extraction: Genomic DNA was extracted from Bonamia sp. infected Ostrea chilensis using the following procedure. Ethanol fixed tissues were suspended in 1 ml of extraction buffer (NaCl 100 mM, EDTA 25 mM, pH 8, SDS 0.5%) with proteina...

The protistan parasite Mikrocytos mackini, the causative agent of Denman Island disease in the oyster Crassostrea gigas in British Columbia, Canada, is of wide concern because it can infect other oyster species and because its life cycle, mode of transmission, and origins are unknown. PCR and fluorescent in situ hybridization (FISH) assays were developed for M. mackini, the PCR assay was validated against standard histopathological diagnosis, and a preliminary phylogenetic analysis of the M. mackini small-subunit ribosomal RNA gene (SSU rDNA) was undertaken. A PCR designed specifically not to amplify host DNA generated a 544 bp SSU rDNA fragment from M. mackini-infected oysters and enriched M. mackini cell isolates, but not from uninfected control oysters. This fragment was confirmed by FISH to be M. mackini SSU rDNA. A M. mackini-specific PCR was then designed which detected 3 to 4 × more M. mackini infections in 1056 wild oysters from Denman Island, British Columbia, than standard histopathology. Mikrocytos mackini prevalence estimates based on both PCR and histopathology increased (PCR from 4.4 to 7.4%, histopathology from 1.2 to 2.1%) when gross lesions were processed in addition to standard samples (i.e. transverse sections for histopathology, left outer palp DNA for PCR). The use of histopathology and tissue imprints plus PCR, and standard samples plus observed gross lesions, represented a 'total evidence' approach that provided the most realistic estimates of the true prevalence of M. mackini. Maximum parsimony and evolutionary distance phylogenetic analyses suggested that M. mackini may be a basal eukaryote, although it is not closely related to other known protistan taxa. KEY WORDS: Denman Island disease · Mikrocytos mackini · PCR validation · Fluorescent in situ hybridization Resale or republication not permitted without written consent of the publisherDis Aquat Org 54: [219][220][221][222][223][224][225][226][227] 2003 (OIE 2000) because it is pathogenic to several oyster species and because its life cycle, mode of transmission, and origins are unknown.Mikrocytos mackini might be understood more thoroughly and managed more effectively if specific molecular diagnostic assays existed to complement conventional histopathological techniques. However, M. mackini small-subunit ribosomal DNA (SSU rDNA) eluded identification for a decade, presumably because 'universal' eukaryotic PCRs preferentially amplified the oyster DNA predominating in host-parasite mixtures, and because pure isolates of M. mackini are not available. We describe here: successful amplification of M. mackini 's SSU rDNA gene using a primer pair designed specifically not to amplify host DNA; proof of the identity of this gene using fluorescent in situ hybridization (FISH); design and validation of a PCR assay specific for M. mackini SSU rDNA; and a preliminary SSU rDNA phylogenetic examination of M. mackini 's affinities. MATERIALS AND METHODSAmplification of presumptive Mikrocytos mackini SSU rDNA. A rapid PCR cloning approa...