Interspecific hybridization in oysters: Restriction Enzyme Digestion Chromosome Banding confirms Crassostrea angulata × Crassostrea gigas F1 hybrids

The taxonomic status of the two commercially important cupped oysters, Crassostrea angulata, the Portuguese oyster (Lamarck, 1819) and Crassostrea gigas, the Japanese oyster (Thunberg, 1793) has long been in question. The recent observation of the hybridization between C. gigas and C. angulata and the production of fertile F1s led us to search for cytogenetic evidence of both parental genomes in the interspecific hybrids. The cytogenetic characterization of the hybrids was performed by the use of restriction endonuclease treatments. This technique has recently shown the potential for individual chromosome identification by banding in oysters. Chromosomes of C. gigas, C. angulata and their hybrids were treated with two different restriction enzymes (ApaI and HaeIII), stained with Giemsa, and examined for banding patterns. These chromosome markers allowed the parental haploid sets to be identified in the hybrids. The analysis of the banded karyotypes of the interspecific hybrids showed that for each chromosome pair, one of the homologues presented a banding pattern consistent with that of C. gigas and the other homologue presented a banding pattern consistent with that of C. angulata. These cytogenetic results substantiate the reported interspecific hybridization between C. gigas and C. angulata. In view of these results and taking into account the present expansion of C. gigas aquaculture in southern Europe, the question of the need for preservation of pure C. angulata stocks should be raised as only a few populations remain in the south of Spain and Portugal. Recently, changes in the genetic composition of populations in southern Portugal have indeed been observed, showing that human activities have created contact zones between the two taxa while no natural sympatric zones exist in Europe.     

Mitochondrial and nuclear DNA phylogeography of Crassostrea angulata, the Portuguese oyster endangered in Europe

The respective status of the Portuguese oyster, Crassostrea angulata, and the Pacific oyster, Crassostrea gigas, has long been a matter of controversy. Morphological and physiological similarities, homogeneity of allozyme allelic frequencies between populations of the two taxa and the demonstration of hybridization lead most authors to suggest that they should be regrouped within the same species. The risk of introgression and the present expansion of C. gigas aquaculture in Europe raises the question of the need for preservation of C. angulata in Europe, as only a few populations remain. We studied European and Asian populations of C. gigas and C. angulata using microsatellite and mitochondrial DNA markers to estimate their genetic diversity and differentiation. The analysis of genetic distances and the distribution of allelic and haplotype frequencies revealed significant genetic differences between taxa, showing two clusters: (1) C. gigas French and Japanese populations and (2) C. angulata Portuguese and Taiwanese populations. The Asian origin of the Crassostrea angulata taxa is therefore confirmed. Unlike previous studies based on allozymes, significant nuclear genome differences were noted between C. angulata and C. gigas. Despite the presumed history of the introduction of C. angulata into Southern Europe, these populations did not show any significant reduction of variability compared to Taiwanese populations. Any conservation plans for European C. angulata populations should take its non-native origin into account. They represent a valuable genetic resources for European breeding program.     

Microsatellite Analysis of 6-Hour-Old Embryos Reveals No Preferential Intraspecific Fertilization Between Cupped Oysters Crassostrea gigas and Crassostrea angulata

Experimental examination of reproductive isolation is the first step in understanding hybridization processes. Here, we studied preferential fertilization between 2 cupped oyster taxa, Crassostrea angulata and Crassostrea gigas, as a potential prezygotic reproductive isolation. Early examination of sperm competition is now possible by molecular analysis of oyster embryos. This avoids the confounding effect of differential mortality during the larval stage. Six hundred embryos were sampled from 2 crosses. Three microsatellite loci were enough to determine without ambiguity the taxa of contributing sires of embryos. No evidence of preferential fertilization between gametes from the same taxa was shown. A significantly higher contribution of the C. gigas males was revealed with the C. angulata females, but not with the C. gigas females, which might suggest early heterosis or interaction differences between gametes. In the light of these results, natural hybridization between both taxa can be expected in cases of their geographical coexistence, as in the Southern European populations in which both taxa are in contact as a result of aquaculture development. Received May 6, 2000; accepted March 6, 2001.     

Molecular and histological identification of Marteilioides infection in Suminoe Oyster Crassostrea ariakensis, Manila Clam Ruditapes philippinarum and Pacific Oyster Crassostrea gigas on the south coast of Korea

The oyster ovarian parasite Marteilioides chungmuensis has been reported from Korea and Japan, damaging the oyster industries. Recently, Marteilioides-like organisms have been identified in other commercially important marine bivalves. In this study, we surveyed Marteilioides infection in the Manila clam Ruditapes philippinarum, Suminoe oyster Crassostrea ariakensis, and Pacific oyster Crassostrea gigas, using histology and Marteilioides-specific small subunit (SSU) rDNA PCR. The SSU rDNA sequence of M. chungmuensis (1716 bp) isolated from C. gigas in Tongyoung bay was 99.9% similar to that of M. chungmuensis reported in Japan. Inclusions of multi-nucleated bodies in the oocytes, typical of Marteilioides infection, were identified for the first time in Suminoe oysters. The SSU rDNA sequence of a Marteilioides-like organism isolated from Suminoe oysters was 99.9% similar to that of M. chungmuensis. Marteilioides sp. was also observed from 7 Manila clams of 1840 individuals examined, and the DNA sequences of which were 98.2% similar to the known sequence of M. chungmuensis. Unlike Marteilioides infection of Pacific oysters, no remarkable pathological symptoms, such as large multiple lumps on the mantle, were observed in infected Suminoe oysters or Manila clams. Distribution of the infected Manila clams, Suminoe oysters and Pacific oysters was limited to small bays on the south coast, suggesting that the southern coast is the enzootic area of Marteilioides infection.     

High survival and growth rates of introduced Pacific oysters may cause restrictions on habitat use by native mussels in the Wadden Sea

Pacific oysters Crassostrea gigas (Thunberg, 1793) were introduced to the northern Wadden Sea (North Sea, Germany) by aquaculture in 1986 and finally became established. Even though at first recruitment success was rare, three consecutive warm summers led to a massive increase in oyster abundances and to the overgrowth of native mussel beds (Mytilus edulis L.). These mussels constitute biogenic reefs on the sand and mud flats in this area. Survival and growth of the invading C. gigas were investigated and compared with the native mussels in order to predict the further development of the oyster population and the scope for coexistence of both species. Field experiments revealed high survival of juvenile C. gigas (approximately 70%) during the first three months after settlement. Survival during the first winter varied between > 90% during a mild and 25% during a cold winter and was independent of substrate (i.e., mussels or oysters) and tide level. Within their first year C. gigas reached a mean length of 35-53 mm, and within two years they grew to 68-82 mm, which is about twice the size native mussels would attain during that time. Growth of juvenile oysters was not affected by substrate (i.e., sand, mussels, and other oysters), barnacle epibionts and tide level, but was facilitated by fucoid algae. By contrast, growth of juvenile mussels was significantly higher on sand flats than on mussel or oyster beds and higher in the subtidal compared to intertidal locations. Cover with fucoid algae increased mussel growth but decreased their condition expressed as dry flesh weight versus shell weight. High survival and growth rates may compensate for years with low recruitment, and may therefore allow a fast population increase. This may lead to restrictions on habitat use by native mussels in the Wadden Sea.     

Trophic interactions between two introduced suspension-feeders, Crepidula fornicata and Crassostrea gigas, are influenced by seasonal effects and qualitative selection capacity

The effects of season and qualitative selection capacity on trophic relationships between two sympatric invasive suspension-feeders, Crepidula fornicata and Crassostrea gigas, were investigated in Bourgneuf Bay (France) from January 2003 to June 2004. Carbon and nitrogen stable isotopic deviations, δ¹³C and δ¹⁵N, of common Atlantic slippersnails and Pacific oysters were analysed relative to isotopic composition and availability of end-members.Slippersnail deviations were less variable over the sampling period compared with those of oysters. Significant differences between δ¹³C and δ¹⁵N of C. fornicata and C. gigas were found from winter to early summer, and linked to major isotopic changes in oysters. We identified three distinct seasonal periods: January to March when oysters were ¹⁵N-enriched compared to slippersnails and to themselves at other times of the year, April to June-July when oysters showed a ¹⁵N-depletion and a more marked ¹³C-depletion compared to slippersnails and to themselves at other times of the year, and July-August to December when both species presented similar carbon and nitrogen deviations. Species-specific differences in qualitative selection capability may explain these seasonal differences in isotopic deviations. Whereas the isotopic composition of the indiscriminate suspension-feeding slippersnails reflects the composition of the seston throughout the year, the oyster is capable of qualitative selection. The oyster isotopic compositions are consistent with a facultative activation of selection mechanisms under conditions of qualitative and quantitative food limitation, notably the preferential ingestion and assimilation of the dominant organic source in the suspended pool.We conclude that C. fornicata and C. gigas are trophic competitors only in winter and spring at this site, where detrital end-members are major POM components. These results underscore (1) the importance of long-term (annual) studies in the evaluation of potential trophic competition, and (2) the necessity to include the qualitative selection capacities of suspension-feeders in future interpretations of trophic relationships in marine coastal ecosystems.     

Microsatellite analysis of gynogenetic families in the Pacific oyster, Crassostrea gigas

Five families of gynogenetic diploid Pacific oyster (Crassostrea gigas) were induced by inhibiting the second polar body in meiotic cell division of eggs fertilized with UV-irradiated sperm. Segregation patterns of eight microsatellite loci were investigated in the gynogenetic diploid offspring; the proportion of heterozygous progeny was used to estimate microsatellite-centromere (M-C) distances. Mendelian inheritance was confirmed for the eight loci by examining the genotypic segregation in the control crosses. Three of the eight microsatellite loci showed the existence of null alleles in four control crosses. All gynogenetic offspring only possessed the alleles of the mother, indicating 100% success level for the five families. The M-C recombination frequency estimates ranged from 0.62 to 0.77 (0.72 mean), comparable to those in the oyster based on allozyme markers and suggesting that meiotic gynogenesis does not appear to be a very efficient inbreeding method in the oyster. Recombination frequencies observed were often higher than the theoretical maximum of 0.67, indicating the existence of positive interference after a single chiasma formation in some chromosomes. Information on the positions of centromeres in relation to the microsatellite loci will represent a contribution toward assembly of genetic maps in C. gigas.     

Specific Detection of Pacific Oyster (Crassostrea gigas) Larvae in Plankton Samples Using Nested Polymerase Chain Reaction

Management of sustainable Pacific oyster fisheries would be assisted by an early, rapid, and accurate means of detecting their planktonic larvae. Reported here is an approach, based on polymerase chain reaction (PCR), for the detection of Pacific oyster larvae in plankton samples. Species-specific primers were designed by comparing partial mitochondrial cytochrome oxidase subunit I (COI) sequences from Crassostrea gigas, with other members of the family Ostreidae including those of Crassostrea angulata. Assay specificity was empirically validated through screening DNA samples obtained from several species of oysters. The assay was specific as only C. gigas samples returned PCR-positive results. A nested PCR approach could consistently detect 5 or more D-hinge-stage larvae spiked into a background of about 146 mg of plankton. The assay does not require prior sorting of larvae. We conclude that the assay could be used to screen environmental and ballast water samples, although further specificity testing against local bivalve species is recommended in new locations.     

A cDNA Microarray for Crassostrea virginica and C. gigas

The eastern oyster, Crassostrea virginica, and the Pacific oyster, C. gigas, are species of global economic significance as well as important components of estuarine ecosystems and models for genetic and environmental studies. To enhance the molecular tools available for oyster research, an international group of collaborators has constructed a 27,496-feature cDNA microarray containing 4460 sequences derived from C. virginica, 2320 from C. gigas, and 16 non-oyster DNAs serving as positive and negative controls. The performance of the array was assessed by gene expression profiling using gill and digestive gland RNA derived from both C. gigas and C. virginica, and digestive gland RNA from C. ariakensis. The utility of the microarray for detection of homologous genes by cross-hybridization between species was also assessed and the correlation between hybridization intensity and sequence homology for selected genes determined. The oyster cDNA microarray is publicly available to the research community on a cost-recovery basis.     

Can bivalve veligers escape feeding currents of adult bivalves?

While the stock of introduced Pacific oysters (Crassostrea gigas) increased in the Oosterschelde estuary (SW Netherlands), so did the filtration pressure of all bivalve species together. In the same period, stocks of native bivalves declined slightly. The expansion of Pacific oysters in Dutch estuaries might be partially due to better abilities of their larvae to avoid or escape filtration, compared to larvae of native bivalves. In this context, escape and swimming abilities of Pacific oyster larvae and the larvae of the native blue mussel (Mytilus edulis) were compared.Swimming behaviour of C. gigas larvae and larvae of M. edulis was recorded in still water and in a suction current mimicking a bivalve feeding current, in a horizontal and in a vertical plane. Larval swimming behaviour in a suction flow field was reconstructed by subtracting local water movement vectors from the total movement of larvae, yielding movement paths due to larval swimming alone.Swimming speeds and the rate of displacement in vertical direction of C. gigas and M. edulis larvae were related to larval shell length, and to the pitch of up- or downward swimming.Larvae of both species did not show escape reactions in a suction flow field. With increasing shell length, larval swimming speeds of both species increased significantly. Swimming speeds of C. gigas larvae were significantly higher than swimming speeds of M. edulis larvae, resulting in a faster vertical displacement. The ability to migrate to more favourable water layers faster may offer C. gigas an advantage over native bivalves with slower swimming larvae.     

盐度对长牡蛎和近江牡蛎及其杂交稚贝生长和存活的影响

2011年8月以长牡蛎自繁组GG(Crassostrea gigas♀×C.gigas♂),近江牡蛎自繁组AA(C.ariakensis♀×C.ariakensis♂)、正交组GA(Crassostrea gigas♀×C.ariakensis♂)、反交组AG(C.ariakensis♀×C.gigas♂)为实验材料,开展了稚贝对盐度的适应性研究。结果发现长牡蛎的最适生存盐度为15—35,最适生长盐度为25—35;近江牡蛎的最适生存盐度为10—25,最适生长盐度为20—25;GA的最适生存盐度为15—30,最适生长盐度为15—30,AG的最适生存盐度为20—30,最适生长盐度为20—25。GG对低盐度敏感,AA对高盐度敏感,AG具有高盐度存活的杂种优势,在盐度30时,中亲杂种优势HG×A为13.32,单亲杂种优势HGA和HAG分别为1.89和27.88,在盐度40时,HAG上升到400,GA和AG都不具有生长优势。杂种稚贝对盐度适应介于双亲之间,且表现出一定程度的父系遗传特点。     

Characterisation of physiological and immunological differences between Pacific oysters (Crassostrea gigas) genetically selected for high or low survival to summer mortalities and fed different rations under controlled conditions

Within the framework of a national scientific program named “MORtalités ESTivales de l'huître creuse Crassostrea gigas” (MOREST), a family-based experiment was developed to study the genetic basis of resistance to summer mortality in the Pacific oyster, Crassostrea gigas. As part of the MOREST project, the second generation of three resistant families and two susceptible families were chosen and pooled into two respective groups: “R” and “S”. These two groups of oysters were conditioned for 6 months on two food levels (4% and 12% of oyster soft-tissue dry weight in algal dry weight per day) with a temperature gradient that mimicked the Marennes-Oléron natural cycle during the oyster reproductive period. Oyster mortality remained low for the first two months, but then rapidly increased in July when seawater temperature reached 19 °C and above. Mortality was higher in “S” oysters than in “R” oysters, and also higher in oysters fed the 12% diet than those fed 4%, resulting in a decreasing, relative order in cumulative mortality as follows; 12% “S” > 12% “R” > 4% “S” > 4% “R”. Although the observed mortality rates were lower than those previously observed in the field, the mortality differential between “R” and “S” oysters was similar. Gonadal development, estimated by tissue lipid content, followed a relative order yielding a direct, positive relationship between reproductive effort and mortality as we reported precedently by quantitative histology. Regarding hemocyte parameters, one of the most striking observations was that reactive oxygen species (ROS) production was significantly higher in “S” oysters than in “R” oysters in May and June, regardless of food level. The absence of known environmental stress under these experimental conditions suggests that the ROS increase in “S” oyster could be related to their higher reproductive activity. Finally, a higher increase in hyalinocyte counts was observed for”S” oysters, compared to “R” oysters, in July, just before mortality. Taken together, our results suggest an association of genetically based resistance to summer mortality, reproductive strategy and hemocyte parameters.     

Phenotypic and genetic consequences of size selection at the larval stage in the Pacific oyster (Crassostrea gigas)

The life histories of oysters in the genus Crassostrea, like those of most marine bivalves, are typified by high fecundity and low survival in nature. Rearing conditions in hatcheries however ensure optimized density, diet, and temperature. Hatcheries are becoming increasingly important for the production of juveniles in aquaculture, and their culture practices often include culling of slow growing larvae to reduce and synchronize the time taken to reach settlement. Because previous studies have found substantial genetic variation for early life developmental traits in Crassostrea gigas, these culling practices are likely to cause highly different selective pressures in hatcheries from those in the natural environment. We studied the phenotypic and genetic impact of such culling practices in a factorial cross between 10 males and 3 females subjected to progressive culling of the smallest 50% of larvae, compared with a non-culled control. Measurements were made on larval growth, survival, time taken to attain pediveliger stage and settlement success. Culling had a larger effect on the variance of these larval traits than on their means. The larvae in culled cultures were approximately 10% larger than those in controls, whereas the coefficient of variation was reduced by 30-40%. Culling also reduced the mean time to settlement by 12% and its variance by 55%. Using a multiplexed set of microsatellite markers to trace parentage, we also estimated the variance in reproductive success in a controlled experiment to quantify the consequences of intensive hatchery rearing practices. We also focused on changes in effective population size and genetic structure over time (and developmental stages). Our results show a loss of genetic diversity following removal of the smallest larvae by culling, as well as temporally varying genetic structure of the larval population. This supports the existence of genetic variability in early life developmental traits in C. gigas. Culling in hatcheries, like size-related selective pressures in the wild, are likely to have a significant genetic impact, through their effects on the timing of settlement.     

Responses of diploid and triploid Pacific oysters Crassostrea gigas to Vibrio infection in relation to their reproductive status

Several Vibrio species are known to be pathogenic to the Pacific oyster Crassostrea gigas. Survival varies according to pathogen exposure and high mortality events usually occur in summer during gametogenesis. In order to study the effects of gametogenetic status and ploidy (a factor known to affect reproduction allocation in oysters) on vibriosis survival, we conducted two successive experiments. Our results demonstrate that a common bath challenge with pathogenic Vibriosplendidus and Vibrio aestuarianus on a mixture of mature, spawning and non-mature oysters can lead to significant mortality. Previous bath challenges, which were done using only non-mature oysters, had not produced mortality. Immunohistochemical analyses showed the affinity of Vibrio for gonadic tissues, highlighting the importance of sexual maturity for vibriosis infection processes in oysters. Mortality rate results showed poor repeatability between tanks, however, in this bath challenge. We then tested a standardized and repeatable injection protocol using two different doses of the same combination of two Vibrio species on related diploid and triploid oysters at four different times over a year. Statistical analyses of mortality kinetics over a 6-day period after injection revealed that active gametogenesis periods correspond to higher susceptibility to vibriosis and that there is a significant interaction of this seasonal effect with ploidy. However, no significant advantage of triploidy was observed. Triploid oysters even showed lower survival than diploid counterparts in winter. Results are discussed in relation to differing energy allocation patterns between diploid and triploid Pacific oysters.     

The density and spatial arrangement of the invasive oyster Crassostrea gigas determines its impact on settlement of native oyster larvae

Understanding how the density and spatial arrangement of invaders is critical to developing management strategies of pest species. The Pacific oyster, Crassostrea gigas, has been translocated around the world for aquaculture and in many instances has established wild populations. Relative to other species of bivalve, it displays rapid suspension feeding, which may cause mortality of pelagic invertebrate larvae. We compared the effect on settlement of Sydney rock oyster, Saccostrea glomerata, larvae of manipulating the spatial arrangement and density of native S. glomerata, and non‐native C. gigas. We hypothesized that while manipulations of dead oysters would reveal the same positive relationship between attachment surface area and S. glomerata settlement between the two species, manipulations of live oysters would reveal differing density‐dependent effects between the native and non‐native oyster. In the field, whether oysters were live or dead, more larvae settled on C. gigas than S. glomerata when substrate was arranged in monospecific clumps. When, however, the two species were interspersed, there were no differences in larval settlement between them. By contrast, in aquaria simulating a higher effective oyster density, more larvae settled on live S. glomerata than C. gigas. When C. gigas was prevented from suspension feeding, settlement of larvae on C. gigas was enhanced. By contrast, settlement was similar between the two species when dead. While the presently low densities of the invasive oyster C. gigas may enhance S. glomerata larval settlement in east Australian estuaries, future increases in densities could produce negative impacts on native oyster settlement. Synthesis and applications: Our study has shown that both the spatial arrangement and density of invaders can influence their impact. Hence, management strategies aimed at preventing invasive populations reaching damaging sizes should not only consider the threshold density at which impacts exceed some acceptable limit, but also how patch formation modifies this.     

Variability of haemocyte and haemolymph parameters in European flat oyster Ostrea edulis families obtained from brood stocks of different geographical origins and relation with infection by the protozoan Bonamia ostreae

A research project to compare productive traits (growth and mortality), disease susceptibility and immune capability between Ostrea edulis stocks was performed. This article reports the results on the immune capability and its relation with infection by the intrahaemocytic protozoan Bonamia ostreae. Four to five oyster spat families were produced from each of four European flat oyster populations (one from Ireland, one from Greece and two from Galicia, Spain) in a hatchery. The spat were transferred to a raft in the Ría de Arousa (Galicia) for on growing for 2 years. Total haemocyte count (THC) and differential haemocyte count (DHC) were estimated monthly through the second year of growing-out. Three types of haemocytes were distinguished: granulocytes (GH), large hyalinocytes (LHH) and small hyalinocytes (SHH). Significant correlations between the mean relative abundance of GH and SHH of the families and the mean prevalence of B. ostreae, the overall incidence of pathological conditions and the cumulative mortality of the families were found; these correlations supported the hypothesis that high %GH and low %SHH would enhance oyster immune ability and, consequently, would contribute to lower susceptibility to disease and longer lifespan. Infection by B. ostreae involved a significant increase of circulating haemocytes, which affected more markedly the LHH type. The higher the infection intensity the higher the %LHH. This illustrates the ability of B. ostreae to modulate the immune responses of the O. edulis to favour its own multiplication. A significant reduction of the phenoloxidase activity in the haemolymph of oysters O. edulis infected by B. ostreae was observed. Nineteen enzymatic activities in the haemolymph of O. edulis and Crassostrea gigas (used as a B. ostreae resistant reference) were measured using the kit api ZYM^®, Biomerieux. Qualitative and quantitative differences in enzyme activities in both haemocyte and plasma fractions between B. ostreae noninfected O. edulis from different origins were recorded. However, no clear positive association between enzyme activity and susceptibility to bonamiosis was found. The only enzyme detected in the resistant species C. gigas that was not found in the susceptible one O. edulis was β-glucosidase (in plasma). B. ostreae infected O. edulis showed significant increase of some enzyme activities and the occurrence of enzymes that were not detected in noninfected oysters. These changes could be due to infection-induced enzyme synthesis by the host or to enzyme synthesis by the parasite.     

Resistance of the Manila clam (Venerupis philippinarum) to infection with Mikrocytos mackini

Manila clams (Venerupis philippinarum) challenged in laboratory trials via bath exposure proved to be resistant to infections with Mikrocytos mackini (protistan parasite of unknown taxonomic affiliation), while Pacific oysters (Crassostrea gigas) challenged simultaneously using identical conditions developed infections. Although M. mackini was detected by a nucleic acid pathogen specific (PCR) assay in 10-30% of the challenged V. philippinarum that were sampled soon after exposure (0-48 h, n = 40), all of the subsequent V. philippinarum (n = 62) sampled 9-17 weeks post-exposure tested negative for M. mackini by PCR assay. Prevalence of infection for the exposed C. gigas (n = 100) during this same period ranged from 50% to 100% by PCR assay. Infection was confirmed in the oysters (58%, n = 60) by a digoxigenin-labelled DNA probe designed to detect M. mackini by in situ hybridization, but M. mackini was not found in any of the exposed Manila clams (n = 63) using this technique.