Cementing mussels to oysters in the pteriomorphian tree: a phylogenomic approach.

Mussels (Mytilida) are a group of bivalves with ancient origins and some of the most important commercial shellfish worldwide. Mytilida consists of approximately 400 species found in various littoral and deep-sea environments, and are part of the higher clade Pteriomorphia, but their exact position within the group has been unstable. The multiple adaptive radiations that occurred within Pteriomorphia have rendered phylogenetic classifications difficult and uncertainty remains regarding the relationships among most families. To address this phylogenetic uncertainty, novel transcriptomic data were generated to include all five orders of Pteriomorphia. Our results, derived from complex analyses of large datasets from 41 transcriptomes and evaluating possible pitfalls affecting phylogenetic reconstruction (matrix occupancy, heterogeneity, evolutionary rates, evolutionary models), consistently recover a well-supported phylogeny of Pteriomorphia, with the only exception of the most complete but smallest data matrix (Matrix 3: 51 genes, 90% gene occupancy). Maximum-likelihood and Bayesian mixture model analyses retrieve strong support for: (i) the monophyly of Pteriomorphia, (ii) Mytilida as a sister group to Ostreida, and (iii) Arcida as sister group to all other pteriomorphians. The basal position of Arcida is congruent with its shell microstructure (solely composed of aragonitic crystals), whereas Mytilida and Ostreida display a combination of a calcitic outer layer with an aragonitic inner layer composed of nacre tablets, the latter being secondarily lost in Ostreoidea.

Richness and distribution of tropical oyster parasites in two oceans.

Parasites can exert strong effects on population to ecosystem level processes, but data on parasites are limited for many global regions, especially tropical marine systems. Characterizing parasite diversity and distributions are the first steps towards understanding the potential impacts of parasites. The Panama Canal serves as an interesting location to examine tropical parasite diversity and distribution, as it is a conduit between two oceans and a hub for international trade. We examined metazoan and protistan parasites associated with ten oyster species collected from both Panamanian coasts, including the Panama Canal and Bocas del Toro. We found multiple metazoan taxa (pea crabs, Stylochus spp., Urastoma cyrinae). Our molecular screening for protistan parasites detected four species of Perkinsus (Perkinsus marinus, Perkinsus chesapeaki, Perkinsus olseni, Perkinsus beihaiensis) and several haplosporidians, including two genera (Minchinia, Haplosporidium). Species richness was higher for the protistan parasites than for the metazoans, with haplosporidian richness being higher than Perkinsus richness. Perkinsus species were the most frequently detected and most geographically widespread among parasite groups. Parasite richness and overlap differed between regions, locations and oyster hosts. These results have important implications for tropical parasite richness and the dispersal of parasites due to shipping associated with the Panama Canal.

Interacting effects of habitat structure and seeding with oysters on the intertidal biodiversity of seawalls.

The construction of artificial structures, such as seawalls, is increasing globally, resulting in loss of habitat complexity and native species biodiversity. There is increasing interest in mitigating this biodiversity loss by adding topographic habitat to these structures, and/or seeding them with habitat-forming species. Settlement tile experiments, comparing colonisation of species to more and less complex habitats, have been used to inform eco-engineering interventions prior to their large-scale implementation. Most studies have focused on applying one type of intervention (either adding habitat structure or seeding with native organisms), so it is unclear whether there are greater benefits to biodiversity when multiple interventions are combined. Using a fully orthogonal experiment, we assessed the independent and interactive effects of habitat structure (flat vs. crevice/ridges) and seeding with native oysters (unseeded vs. seeded) on the biodiversity of four different functional groups (sessile and mobile taxa, cryptobenthic and pelagic fishes). Concrete tiles (flat unseeded, flat seeded, complex unseeded and complex seeded) were deployed at two sites in Sydney Harbour and monitored over 12 months, for the survival and colonisation of oysters and the species density and abundances of the four functional groups. The survival of seeded oysters was greater on the complex than flat tiles, at one of the two sites, due to the protective role of crevices. Despite this, after 12 months, the species density of sessile invertebrates and the percentage cover of seeded and colonising oysters did not differ between complex and seeded tiles each of which supported more of these variables than the flat unseeded tiles. In contrast, the species density of mobile invertebrates and cryptobenthic fishes and the MaxN of pelagic fishes, at 1 month, were only positively influenced by seeding with oysters, which provided food as well as habitat. Within the complex seeded and unseeded tiles, there was a greater species density of sessile taxa, survival and percentage cover of oysters in the crevices, which were more humid and darker at month 12, had lower high temperature extremes at months 1 and 12, than on the ridges or flat tiles. Our results suggest that eco-engineering projects which seek to maximise the biodiversity of multiple functional groups on seawalls, should apply a variety of different microhabitats and habitat-forming species, to alter the environmental conditions available to organisms.

Differential expression of genes encoding anti-oxidant enzymes in Sydney rock oysters, Saccostrea glomerata (Gould) selected for disease resistance.

Sydney rock oysters (Saccostrea glomerata) selectively bred for disease resistance (R) and wild-caught control oysters (W) were exposed to a field infection of disseminating neoplasia. Cumulative mortality of W oysters (31.7%) was significantly greater than R oysters (0.0%) over the 118 days of the experiment. In an attempt to understand the biochemical and molecular pathways involved in disease resistance, differentially expressed sequence tags (ESTs) between R and W S. glomerata hemocytes were identified using the PCR technique, suppression subtractive hybridisation (SSH). Sequencing of 300 clones from two SSH libraries revealed 183 distinct sequences of which 113 shared high similarity to sequences in the public databases. Putative function could be assigned to 64 of the sequences. Expression of nine ESTs homologous to genes previously shown to be involved in bivalve immunity was further studied using quantitative reverse-transcriptase PCR (qRT-PCR). The base-line expression of an extracellular superoxide dismutase (ecSOD) and a small heat shock protein (sHsP) were significantly increased, whilst peroxiredoxin 6 (Prx6) and interferon inhibiting cytokine factor (IK) were significantly decreased in R oysters. From these results it was hypothesised that R oysters would be able to generate the anti-parasitic compound, hydrogen peroxide (H(2)O(2)) faster and to higher concentrations during respiratory burst due to the differential expression of genes for the two anti-oxidant enzymes of ecSOD and Prx6. To investigate this hypothesis, protein extracts from hemolymph were analysed for oxidative burst enzyme activity. Analysis of the cell free hemolymph proteins separated by native-polyacrylamide gel electrophoresis (PAGE) failed to detect true superoxide dismutase (SOD) activity by assaying dismutation of superoxide anion in zymograms. However, the ecSOD enzyme appears to generate hydrogen peroxide, presumably via another process, which is yet to be elucidated. This corroborates our hypothesis, whilst phylogenetic analysis of the complete coding sequence (CDS) of the S. glomerata ecSOD gene is supportive of the atypical nature of the ecSOD enzyme. Results obtained from this work further the current understanding of the molecular mechanisms involved in resistance to disease in this economically important bivalve, and shed further light on the anomalous oxidative processes involved.

Mass spectrometry based sensor strategies for the authentication of oysters according to geographical origin.

This study was undertaken to investigate the relevance of using the pyrolysis-MS (Py-MS) technique to discriminate the production area of oysters harvested over two years and to assess from the data of the second year of harvest the potential of an alternative MS-based technique, the solid phase microextraction-MS (SPME-MS), to perform this discrimination. Oysters were harvested in various areas of France, and models of discrimination according to harvest season were built from Py-MS fingerprints and from virtual SPME-MS fingerprints obtained by summing the mass spectra generated by the SPME-GC-MS system. The treatment of the Py-MS data by a 21-12-3 artificial neural networks led to a correct classification of only 89.2% of the oyster samples according to shoreline. The misclassifications thus did not allow use of the Py-MS technique as a relevant tool for authentication of oyster origin. The assessment of the potential of the virtual SPME-MS fingerprints to discriminate the production area of oysters was undertaken on a part of the sample set. The virtual SPME-MS data were pretreated according to two methods, filtering of raw data (FRD) and comprehensive combinatory standard correction (CCSC), a recently developed chemometric method used for the correction of instrumental signal drifts in MS systems. The results obtained with the virtual SPME-MS fingerprints are promising because this technique, when the data were pretreated by the CCSC method, led to a successful discrimination of the oyster samples not only according to shoreline but also according to production region. This study confirms that an efficient correction method (CCSC) of instrumental drifts can considerably increase the discriminative information contained in the volatile fraction of food products.

Genome-wide comparisons reveal evidence for a species complex in the black-lip pearl oyster Pinctada margaritifera (Bivalvia: Pteriidae).

Evolutionary relationships in the black-lip pearl oyster Pinctada margaritifera which is highly valued for pearl production remain poorly understood. This species possesses an 18,000 km Indo-Pacific natural distribution, and its current description includes six subspecies defined exclusively on morphological characters. To evaluate its taxonomic identity using molecular data, 14 populations in both the Indian and Pacific Oceans (n = 69), and the congeneric taxa P. maxima and P. mazatlanica (n = 29 and n = 10, respectively) were sampled. Phylogenomic reconstruction was carried out using both 8,308 genome-wide SNPs and 10,000 dominant loci (DArTseq PAVs). Reconstructions using neighbour-joining (Nei's 1972 distances), maximum likelihood and Bayesian approaches all indicate that the taxonomy of P. margaritifera is quite complex, with distinct evolutionary significant units (ESUs) identified within Tanzanian and Iranian populations. Contrastingly, phylogenies generated for Pacific Ocean oysters resolved a large monophyletic clade, suggesting little support for two current morphological subspecies classifications. Furthermore, P. mazatlanica formed a basal clade closest to French Polynesian P. margaritifera, suggesting it may be conspecific. Collectively, these findings provide evidence that P. margaritifera comprises a species complex, perhaps as a result of population fragmentation and increased divergence at range limits.

Comparative De Novo transcriptome analysis of the Australian black-lip and Sydney rock oysters reveals expansion of repetitive elements in Saccostrea genomes.

Ostreid oysters (the 'true oysters') represent a large and commercially important family of bivalve molluscs. Several species, such as the Pacific oyster (Magallana gigas), the American oyster (Crassostrea virginica), the European oyster (Ostrea edulis) and the Sydney rock oyster (Saccostrea glomerata), are currently farmed at a large scale. However a number of other species may also be suitable for commercial-scale aquaculture. One such species is the 'black-lip oyster', a large Saccostrea species of uncertain taxonomic affinity found in northern Australia. Here, phylogenetic analysis of the COI gene places this oyster within a clade identified in a previous study of Japanese Saccostrea species, 'Saccostrea lineage J'. To facilitate comparisons between this oyster and the better-studied S. glomerata, de novo transcriptomes were generated from larval stages and adult tissues of both species. Patterns of orthology indicated an expansion of repetitive elements within Saccostrea genomes when compared to M. gigas and C. virginica, which may be reflected in increased evolutionary rates and/or genome sizes. The generation of high-quality transcriptomes for these two commercially relevant oysters provides a valuable resource for gene identification and comparison of molecular processes in these and other mollusc species.

netview p: a network visualization tool to unravel complex population structure using genome-wide SNPs.

Network-based approaches are emerging as valuable tools for the analysis of complex genetic structure in wild and captive populations. netview p combines data quality control with the construction of population networks through mutual k-nearest neighbours thresholds applied to genome-wide SNPs. The program is cross-platform compatible, open-source and efficiently operates on data ranging from hundreds to hundreds of thousands of SNPs. The pipeline was used for the analysis of pedigree data from simulated (n = 750, SNPs = 1279) and captive silver-lipped pearl oysters (n = 415, SNPs = 1107), wild populations of the European hake from the Atlantic and Mediterranean (n = 834, SNPs = 380) and grey wolves from North America (n = 239, SNPs = 78 255). The population networks effectively visualize large- and fine-scale genetic structure within and between populations, including family-level structure and relationships. netview p comprises a network-based addition to other population analysis tools and provides user-friendly access to a complex network analysis pipeline through implementation in python.

Massive expansion and functional divergence of innate immune genes in a protostome.

The molecules that mediate innate immunity are encoded by relatively few genes and exhibit broad specificity. Detailed annotation of the Pacific oyster (Crassostrea gigas) genome, a protostome invertebrate, reveals large-scale duplication and divergence of multigene families encoding molecules that effect innate immunity. Transcriptome analyses indicate dynamic and orchestrated specific expression of numerous innate immune genes in response to experimental challenge with pathogens, including bacteria, and a pathogenic virus. Variable expression of individual members of the multigene families encoding these genes also occurs during different types of abiotic stress (environmentally-equivalent conditions of temperature, salinity and desiccation). Multiple families of immune genes are responsive in concert to certain biotic and abiotic challenges. Individual members of expanded families of immune genes are differentially expressed under both biotic challenge and abiotic stress conditions. Members of the same families of innate immune molecules also are transcribed in developmental stage- and tissue-specific manners. An integrated, highly complex innate immune system that exhibits remarkable discriminatory properties and responses to different pathogens as well as environmental stress has arisen through the adaptive recruitment of tandem duplicated genes. The co-adaptive evolution of stress and innate immune responses appears to have an ancient origin in phylogeny.

The molecular phylogeny of oysters based on a satellite DNA related to transposons.

We have analysed a centromeric satellite DNA family that is conserved in several commercial and non-commercial oyster species (Ostrea edulis, O. stentina, Crassostrea angulata, C. gigas, C. gasar, C. ariakensis, C. virginica and C. sikamea). This satellite DNA family is composed of AT-rich repeat sequences of 166+/-2 bp and presents a 9-bp motif similar to the mammalian CENP-B box. The homology of oyster HindIII satellite DNA with satellite DNAs from other bivalves and its relation to a part of a mobile element suggest the existence of an ancient transposable element as a generating unit of satellite DNA in bivalve molluscs. Taking advantage of its degree of conservation in oyster species, we have used this element as a taxonomic marker. This marker clearly supports a high degree of differentiation between O. edulis and O. stentina, and, conversely, upholds the contention that C. gigas and C. angulata are the same species. Finally, we have used HindIII satellite DNA as a phylogenetic marker between these species, revealing two clades, one formed by Asiatic species (C. angulata, C. gigas and C. ariakensis) and another by the European, American and African species (O. edulis, C. virginica and C. gasar, respectively).

Molecular genetic identification tools for three commercially cultured oysters (Crassostrea belcheri, Crassostrea iredalei, and Saccostrea cucullata) in Thailand.

Molecular genetic keys for identification of 3 commercially cultured oysters (Crassostrea belcheri, Crassostrea iredalei, and Saccostrea cucullata) in Thailand were developed based on restriction analysis of 18S ribosomal DNA and cytochrome oxidase subunit I (COI). Digestion of the amplified 18S rDNA with Hinf I unambiguously differentiated Crassostrea oysters from Saccostrea oysters and Striostrea (Parastriostrea) mytiloides. In addition, species-specific restriction fragment length polymorphism patterns of C. belcheri, C. iredalei, and S. cucullata were consistently observed when the gel-eluted COI was digested with Mbo I and Dde I. Thirty composite haplotypes were observed across all individuals. Species-specific composite haplotypes were found in C. belcheri (AAAA and AAAB), C. iredalei (AABC and AABU), and S. cucullata (BBCD and BBCE), respectively. The most common composite haplotype of COI in C. belcheri (AAAA), C. iredalei (AABC), and S. cucullata (BBCD) was amplified, cloned, and sequenced. Detection of C. belcheri and C. iredalei based on polymerase chain reaction was further developed using more specific primers (HCO2198 and R372) followed by digestion of a 372-bp product with Mbo I.

Genetic relationship between cultured populations of Pacific oyster revealed by RAPD analysis.

We developed random amplified polymorphic DNA (RAPD) analysis for the assessment of the genetic relationship between cultured populations of the Pacific oyster Crassostrea gigas Thunberg in Hiroshima and Goseong, the largest oyster farming areas in Japan and Korea, respectively. Of 25 arbitrary primers comprising decamer nucleotides of random sequences, polymerase chain reaction amplifications with 5 different primers gave reproducible electrophoretic patterns. A total of 49 RAPD markers were clearly identified for the Hiroshima and Goseong populations, and 46 markers were polymorphic presenting mean polymorphism rates of the respective populations at 92.29% and 93.32%. Pairwise genetic distances of each 20 individuals from these populations served to produce a UPGMA dendrogram. The dendrogram comprised two main clusters, one of which was a nested cluster including all individuals of the Hiroshima population along with 12 individuals of the Goseong population, and the other cluster included the remaining individuals of the Goseong population. Results indicate that RAPD markers are useful for the assessment of the genetic relationships between populations of the Pacific oyster and further that a significant portion of oysters imported from Korea could be genetically related to the Hiroshima population.

Trans-atlantic distribution of a mangrove oyster species revealed by 16S mtDNA and karyological analyses.

Three species of mangrove oysters, Crassostrea rhizophorae, C. brasiliana, and C. gasar, have been described along the Atlantic shores of South America and Africa. Because the distribution of these molluscs is of great biological and commercial interest, their taxonomy and distribution deserve further clarification. Therefore, 15 populations were sampled from both continents. Their 16S mitochondrial polymorphism was studied by sequencing and PCR-RFLP analysis. Two haplotypes were identified. Haplotype a was the only one observed in Africa, but it was also observed in South America together with haplotype b. Because C. gasar is the only mangrove oyster identified on the west coast of Africa, haplotype a was attributed to this species, which has thus been shown to occur in South America. Haplotype b is attributed to C. rhizophorae. The karyotypes of specimens of C. gasar, from Africa and from South America, were very similar, and both species were observed at the same location in Brazil. The occurrence of C. gasar in South America adds a third species-in addition to C. rhizophorae and C. brasiliana-to the list of species present along these coasts. The predominant surface circulation patterns in this part of the Atlantic Ocean favor the hypothesis that C. gasar was transported from Africa to America. Finally, a phylogenetic tree built with seven 16S sequences from Crassostrea and Saccostrea species showed that C. gasar is intermediate between the American Crassostrea species (C. virginica and C. rhizophorae) and the Asian species (C. gigas and C. ariakensis).

[Electrophoretic study of gene-enzyme systems in oysters classified as Crassostrea gigas (Thunberg, 1793) and Crassostrea angulata (Lamarck, 1819) (Mollusca: Ostreidae)]. / Studio elettroforetico dei sistemi gene-enzima in ostriche classificate come Crassostrea gigas (Thunberg, 1793) e Crassostrea angulata (Lamarck, 1819) (Mollusca: Ostreidae).

As a further contribution to the systematics of species belonging to the genus Crassostrea, electrophoretic studies of gene-enzyme system have been carried out on five populations of oysters regarded as either Crassostrea gigas or Crassostrea angulata species. Two populations of C. gigas from Japan (Karakawa and Hiroshima), one of C. gigas from France (Fouras), one of C. angulata from Portugal (Sado) and one of Crassostrea sp. from Italy (Chioggia) have been examined. The electrophoretic study of 20 enzyme loci and the genetic distance value showed no significant genetic divergence among the Crassostrea sp. populations. Thus the results of this study seem to confirm and extend to other populations what obtained by Buroker et al. (1979), that is a remarkable genetical identity between the two species Crassostrea gigas and Crassostrea angulata.

Genomic resource development for shellfish of conservation concern.

Effective conservation of threatened species depends on the ability to assess organism physiology and population demography. To develop genomic resources to better understand the dynamics of two ecologically vulnerable species in the Pacific Northwest of the United States, larval transcriptomes were sequenced for the pinto abalone, Haliotis kamtschatkana kamtschatkana, and the Olympia oyster, Ostrea lurida. Based on comparative species analysis the Ostrea lurida transcriptome (41 136 contigs) is relatively complete. These transcriptomes represent the first significant contribution to genomic resources for both species. Genes are described based on biological function with particular attention to those associated with temperature change, oxidative stress and immune function. In addition, transcriptome-derived genetic markers are provided. Together, these resources provide valuable tools for future studies aimed at conservation of Haliotis kamtschatkana kamtschatkana, Ostrea lurida and related species.

Evolutionary patterns in pearl oysters of the genus Pinctada (Bivalvia: Pteriidae).

Pearl oysters belonging to the genus Pinctada (Bivalvia: Pteriidae) are widely distributed between the Indo-Pacific and western Atlantic. The existence of both widely distributed and more restricted species makes this group a suitable model to study diversification patterns and prevailing modes of speciation. Phylogenies of eight out of the 11 currently recognised Pinctada species using mitochondrial (cox1) and nuclear (18S rRNA) data yielded two monophyletic groups that correspond to shell size and presence/absence of hinge teeth. Character trace of these morphological characters onto the molecular phylogeny revealed a strong correlation. Pinctada margaritifera appears polyphyletic with specimens from Mauritius grouping in a different clade from others of the French Polynesia and Japan. Hence, P. margaritifera might represent a species complex, and specimens from Mauritius could represent a different species. Regarding the putative species complex Pinctada fucata/Pinctada martensii/Pinctada radiata/Pinctada imbricata, our molecular analyses question the taxonomic validity of the morphological characters used to discriminate P. fucata and P. martensii that exhibited the lowest genetic divergence and are most likely conspecific as they clustered together. P. radiata and P. imbricata were recovered as monophyletic. The absence of overlapping distributions between sister lineages and the observed isolation by distance suggests that allopatry is the prevailing speciation mode in Pinctada. Bayesian dating analysis indicated a Miocene origin for the genus, which is consistent with the fossil record. The northward movement of the Australian plate throughout the Miocene played an important role in the diversification process within Pinctada.

Benthic impacts of intertidal oyster culture, with consideration of taxonomic sufficiency.

An investigation of the impacts from elevated intertidal Pacific oyster culture in a New Zealand estuary showed enhanced sedimentation beneath culture racks compared with other sites. Seabed elevation beneath racks was generally lower than between them, suggesting that topographic patterns more likely result from a local effect of rack structures on hydrodynamic processes than from enhanced deposition. Compared with control sites, seabed sediments within the farm had a greater silt/clay and organic content, and a lower redox potential and shear strength. While a marked trend in macrofaunal species richness was not evident, species composition and dominance patterns were consistent with a disturbance gradient, with farm effects not evident 35 m from the perimeter of the racks. Of the environmental variables measured, sediment shear strength was most closely associated with the distribution and density of macrofauna, suggesting that human-induced disturbance from farming operations may have contributed to the biological patterns. To evaluate the taxonomic sufficiency needed to document impacts, aggregation to the family level based on Linnean classification was compared with an aggregation scheme based on ;general groups' identifiable with limited taxonomic expertise. Compared with species-level analyses, spatial patterns of impact were equally discernible at both aggregation levels used, provided density rather than presence/absence data were used. Once baseline conditions are established and the efficacy of taxonomic aggregation demonstrated, a ;general group' scheme provides an appropriate and increasingly relevant tool for routine monitoring.

Phylogenetic analysis of the subclass Pteriomorphia (Bivalvia) from mtDNA COI sequences.

Phylogenetic relationships within the subclass Pteriomorphia (Bivalvia) were examined using sequences of the mitochondrial cytochrome c oxidase subunit I gene. The resultant Minimum Evolution phylogenetic tree strongly supports the existing superfamily-level classification with all the members of each superfamily forming clades. At the same time, it is suggested that: (1) Ostreoidea shows a closer relationship to Pinnoidea and Pterioidea than to the other superfamilies; (2) Pectinoidea, Anomioidea, and Limoidea form a clade, (3) Arcoidea and Limopsoidea form a clade; (4) The subclass Anomalodesmata is closer to the subclass Heterodonta than to Mytiloidea; and (5) The subclass Pteriomorphia is monophyletic. Taking these results as well as published data for nuclear 18S ribosomal DNA (18S rDNA) and Myosin analyses into consideration, a new order-level classification system for Pteriomorphia is proposed.

Supplementation of Perkinsus marinus cultures with host plasma or tissue homogenate enhances their infectivity.

The protozoan oyster parasite Perkinsus marinus can be cultured in vitro in a variety of media; however, this has been associated with a rapid attenuation of infectivity. Supplementation of defined media with products of P. marinus-susceptible (Crassostrea virginica) and -tolerant (Crassostrea gigas, Crassostrea ariakensis) oysters alters proliferation and protease expression profiles and induces differentiation into morphological forms typically seen in vivo. It was not known if attenuation could be reversed by host extract supplementation. To investigate correlations among these changes as well as their association with infectivity, the effects of medium supplementation with tissue homogenates from both susceptible and tolerant oyster species were examined. The supplements markedly altered both cell size and proliferation, regardless of species; however, upregulation of low-molecular-weight protease expression was most prominent with susceptible oysters extracts. Increased infectivity occurred with the use of oyster product-supplemented media, but it was not consistently associated with changes in cell size, cell morphology, or protease secretion and was not related to the susceptibility of the oyster species used as the supplement source.