Individual relationship between aneuploidy of gill cells and growth rate in the cupped oysters Crassostrea angulata, C. gigas and their reciprocal hybrids

The Portuguese oyster, Crassostrea angulata, is taxonomically close to the Pacific oyster, C. gigas, but there are clear genetic and phenotypic differences between these taxa. Among those differences, the faster growth of C. gigas compared with C. angulata has often been observed in the field. Crosses between C. angulata and C. gigas were performed to investigate the relationship between growth variation and somatic aneuploidy at the individual level in the two taxa and their reciprocal hybrids. The different progenies were reared in Ria Formosa (Portugal) under standard farming conditions. Growth rate and survival were significantly higher in C. gigas than in C. angulata, and the hybrids showed intermediate performances. Significant differences were also observed in the proportion of aneuploid cells (PAC) and of missing chromosomes (PMC) between the two taxa, C. angulata showing the highest values. Intermediate values of PAC and PMC were observed in the hybrids, supporting additive genetic bases of these parameters. Our results also confirm the negative correlation between somatic aneuploidy and growth rate at the individual level, as previously reported in C. gigas.     

香港巨牡蛎与长牡蛎种间配子兼容性

为了评估香港巨牡蛎与长牡蛎的配子兼容性,于2010年7月,以性腺成熟的香港巨牡蛎和长牡蛎为材料,研究了环境因子温度(16、20、24、28、32℃)、盐度(10、15、20、25、30)及内在因子精子浓度(100、101、102、103、104个/μL)、个体差异(240次杂交实验)对其受精率的影响,并计算了以上条件下的F50临界值。结果表明:香港巨牡蛎卵子可以与长牡蛎精子受精,但相反方向不能受精,存在着单向受精现象。温度是影响长牡蛎配子兼容性主要因子,盐度是影响香港巨牡蛎的主要因子,而温度、盐度交互作用是影响种间配子兼容性的最主要因子。种间配子温度、盐度的F50临界值分别为20.90℃、14.7。精子浓度及单对杂交中的个体差异均显著影响配子兼容性,精子浓度的F50临界值为2.40个/μL;在240次杂交实验中,有91次受精率不足50%,说明个体差异比例的F50临界值37.92%。从其内在因子来看,长牡蛎平均受精率最高(96.83%),配子兼容性较好;对于香港巨牡蛎(70.98%)与种间配子(58.70%)而言,受精水平相对较低,且存在着较大的个体差异。     

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.     

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.     

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.     

Comparative cryopreservation study of trochophore larvae from two species of bivalves: Pacific oyster (Crassostrea gigas) and Blue mussel (Mytilus galloprovincialis)

Oysters and mussels are among the most farmed species in aquaculture industry around the world. The aim of this study was to test the toxicity of cryoprotective agents to trochophore larvae from two different species of bivalves and develop an improved cryopreservation protocol to ensure greater efficiency in the development of cryopreserved trochophores (14 h old oyster larvae and 20 h old mussel larvae) to normal D-larvae for future developments of hatchery spat production. The cryopreservation protocol producing the best results for oyster trochophores (60.0 ± 6.7% normal D-larvae) was obtained by holding at 0 °C for 5 min then cooling at 1 °C min⁻¹ to −10 °C and holding for 5 min before cooling at 0.5 °C to −35 °C, holding 5 min and then plunging into liquid nitrogen (LN), using 10% ethylene glycol. For mussel experiments, no significant differences were found when cooling at 0.5 °C min⁻¹ or at 1 °C min⁻¹ for CPA combinations with 10% ethylene glycol and at 0.5 °C min⁻¹. Using these combinations, around half of trochophores were able to develop to normal D-larvae post-thawing (48.9 ± 7.6% normal D-larvae).     

香港巨牡蛎和长牡蛎幼虫及稚贝的表型性状

为了评估香港巨牡蛎和长牡蛎在北方沿海的早期表型性状,于2010年7月,以2009年6月在青岛繁育的两种牡蛎为材料,在大连研究了温度(Mt:(22±1.0)℃及Ht:(28±1.0)℃)、盐度(S₂₀:20±1.0及S₃₀:30±1.0)及中间育成环境(ID:室内及OD:室外)对两种牡蛎幼虫及稚贝表型性状的影响。结果表明:香港巨牡蛎壳宽显著大于长牡蛎(P<0.05),壳高及怀卵量显著小于长牡蛎(P<0.05),壳长、鲜重及壳重两者间无显著差异(P>0.05)。在温度和盐度相同情况下,长牡蛎卵径、受精率、孵化率及D形幼虫均大于香港巨牡蛎;香港巨牡蛎幼虫浮游前期生长较慢,而后快于长牡蛎。两种牡蛎幼虫存活能力在15日龄时高温组>中温组;相同温度下,香港巨牡蛎中盐组>高盐组,长牡蛎高盐组>中盐组。幼虫变态期间,较低的温度延迟了变态时间,降低了变态率,使得两种幼虫变态规格大型化。温度是影响幼虫生长、存活、变态的最主要因素,其次为盐度,交互作用几乎尚未起到作用。中间育成阶段,室外比室内培育效果更好,且香港巨牡蛎稚贝壳高在60日龄以后显著大于长牡蛎(P<0.05),环境是影响稚贝生长的最主要因素;无论室内还是室外两种牡蛎稚贝的存活率均在90%以上,且各实验组间无显著差异(P>0.05)。     

Changes in biochemical and hemocyte parameters of the Pacific oysters Crassostrea gigas fed T-Iso supplemented with lipid emulsions rich in eicosapentaenoic acid

The aim of this study was to assess the effect of dietary eicosapentaenoic acid (20:5n-3) on hemocyte parameters such as hemocyte concentration, phagocytosis, and non-stimulated reactive oxygen species (ROS) production in Pacific oysters Crassostrea gigas, as well on proximate biochemical and fatty acid compositions. One-year-old oysters (C. gigas) were fed T-Isochrysis aff. galbana (T-Iso), which is low in 20:5n-3, either alone or with supplements of a lipid emulsion rich in 20:5n-3 at 1%, 10% or 50% (dry weight of the algal ration) for up to 7 weeks. Changes in gill fatty acid composition demonstrated that the lipid emulsion was well ingested by oysters during the dietary conditioning. Biochemical analysis indicated that oysters fed supplements of 50% and, to a lesser extent, 10% lipid emulsions had a higher total lipid content compared with oysters fed other diets, suggesting a more advanced reproductive status for the oysters fed high doses of lipid emulsion. Moreover, some oysters in these two treatment groups spawned during the last three weeks of the seven-week feeding experiment. Lipid supplements had a significant influence on hemocyte concentration, phagocytic index and non-stimulated hemocyte ROS production. After 4 weeks, highest hemocyte concentrations were found in oysters fed on a supplement of 50% lipid emulsion compared with those fed on other diets but the hemocytes derived from these oysters had the lowest short-term phagocytic index. After 7 weeks of dietary conditioning, the ROS production in non-stimulated hemocytes of oysters fed 10% and 50% lipid emulsion declined. These results suggested that 20:5n-3, and perhaps its eicosanoid metabolites, affected oyster hemocyte functions; however, the reproductive status of oysters may also have interfered with the 20:5n-3 dietary effect.     

Detection of white spot syndrome virus (WSSV) in the Pacific oyster Crassostrea gigas

Oysters Crassostrea gigas were placed at water supply canals of three shrimp farms in Guasave, Mexico where WSSV outbreaks occur. Animals were sampled through April-August and September-December to detect WSSV DNA.By using three different PCR protocols, only oysters from a farm undergoing a WSSV outbreak were found WSSV-positive in gills and digestive gland. Two WSSV amplicons were sequenced and they corresponded over 99% to WSSV genome segments. Results showed that oysters can capture WSSV particles suspended in water. Susceptibility of oysters to WSSV infection and their role as a carrier remain to be determined.     

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.     

Carbon and nitrogen relationships in the feeding and growth of the Pacific oyster, Crassostrea gigas (Thunberg)

Bivalve molluscs, in common with consumers in general, use behavioral and physiological mechanisms to balance metabolic requirements with available nutrients. This study considered how the Pacific oyster, Crassostrea gigas, meets the demands of growth and maintenance, measured in terms of carbon and nitrogen, in a variable food environment. Stoichiometry theory helped to evaluate: a) whether feeding behaviour modifies the intake of C and N given seasonal variability in food quality: b) how rates of metabolism and excretion, and C and N growth efficiencies, respond to mismatch between nutrient intake and the oysters' needs. Two field experiments in the Port Stephens estuary, near Sydney, Australia, measured feeding behaviour, metabolic and growth rates relative to seasonal changes in food supply. In a laboratory experiment, relationships between physiological rates and growth were measured to test a model of growth as a function of absorption of C and N. Potential metabolic targets for compensation were the C/N ratios of body tissues, maintenance and/or of soft tissue added as growth. C/N of whole soft issues varied little during the year (mean 5.4). In July (a time of low food availability of poor quality) growth was negligible and the C/N (maintenance) target was 6.7. In March (abundant food of high quality) growth was rapid with a high N-demand; the C/N of growth was 3.9. In November (medium food quality) there was an enhanced C-demand for glycogen storage; the C/N of growth was 7.9. Feeding behaviour changed the balance between C and N intake across months, primarily due to changes in the selection efficiency for nitrogen, which was highest at low filtration rates on particles of high C/N ratio. Nitrogen intake was favoured over C in July. In November, C-intake increased relative to N. In March, when abundant food nitrogen coincided with a high demand for growth, feeding behaviour was neutral with respect to C/N ratios. In all cases C/N of absorbed matter was greater than the C/N of growth. Growth efficiencies for carbon declined with increased C/N of ingested matter due to higher metabolic increments (SDA) when feeding on lower food quality; the metabolic costs of growth did not vary. In contrast, growth efficiencies for nitrogen did not alter with C/N for ingested matter, due in part to increased nitrogen losses, relative to tissue nitrogen content, when feeding on low C/N food. Nitrogen was therefore conserved metabolically relative to C. Both feeding and metabolic processes contributed to compensation for the mismatch between seasonally variable food quality and the demands of growth.     

Changes in motility, ATP content, morphology and fertilisation capacity during the movement phase of tetraploid Pacific oyster (Crassostrea gigas) sperm

Changes in sperm features during the movement phase are especially interesting to study in external fertilization species whose sperm duration movement is long because this implies a significant adaptation of moving cells to the external medium. This study describes the changes in tetraploid Pacific oyster sperm characteristics in relation to time post activation.Sperm individually collected on three tetraploid males were activated in seawater. Their features were analysed over a 24 h period and compared to a sperm pool collected on three diploid males as a reference. The percentage of motile spermatozoa, the intracellular ATP content, and the fine structure of spermatozoa were studied in relation to time post activation. Furthermore, the fertilisation capacity of sperm individually collected on five diploid males was assessed after 1 and 24 h post activation.A forward progressive movement was maintained for at least a 20 h duration. Compared to diploid males, the percentage of motile spermatozoa was lower in tetraploid males. The intracellular ATP concentration was higher in spermatozoa from tetraploid males than in spermatozoa from diploid males. A decrease in ATP content was observed in the first 6 h post activation and severe alterations were observed in sperm morphology after 24 h. Then, a lower fertilisation capacity of sperm from diploid males was observed at the end of the movement phase.The cessation of Pacific oyster sperm motility was unlikely caused by ATP consumption as ATP concentration was still high at the end of sperm movement but rather caused by drastic changes in sperm morphology. Compared to sperm collected on diploid males, the lower quality of sperm from tetraploid males was emphasized by a shorter movement duration and deeper morphological alterations at the end of the movement phase.     

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.     

The effects of intertidal air exposure on the respiratory physiology and the killing activity of hemocytes in the pacific oyster, Crassostrea gigas (Thunberg)

The occurrence of summer mortalities of the commercially important Pacific oyster, Crassostrea gigas, has increased in recent years. These mortality events occur during the late summer when water temperatures are at their highest. Many theories have been proposed concerning the causes including reproductive stress, environmental stress, disease, or synergistic interactions of these factors. C. gigas are grown intertidally and are exposed to the air (emersed) for hours at a time. These organisms can experience extreme changes in temperature during the course of a day. An oyster closed during emersion depletes the oxygen stores to near zero within the shell and builds up CO₂ causing a decrease in tissue pH. The focus of this study is to determine the respiratory (pH, Po₂, Pco₂ and total CO₂) and immune responses of oysters exposed to air at normal seasonal temperatures, and to determine whether these stresses associated with emersion inhibit the immune system of the oyster and contribute to the summer mortalities. The respiratory variables of the hemolymph of oysters submerged at 18 °C (pH = 7.52 ± 0.04 S.E.M., Po₂ = 7.09 ± 0.53 S.E.M. kPa and Pco₂ = 0.20 ± 0.03 S.E.M. kPa) varied significantly from oysters emersed for four hours at 22°C (pH = 7.11 ± 0.03 S.E.M., Po₂ = 3.83 ± 0.15 S.E.M. kPa, Pco₂ = 0.36 ± 0.03 S.E.M. kPa) and those emersed for four hours at 30 °C (pH = 6.84 ± 0.02 S.E.M., Po₂ = 3.10 ± 0.12 S.E.M. kPa, Pco₂ = 1.31 ± 0.06 S.E.M. kPa). The ability of hemocytes to kill the bacterium Vibrio campbellii was assessed using an in vitro assay to generate a killing index. There was no significant difference in the killing index between pH treatment groups (p = 0.856): at pH 7.6 killing index = 50.2% ± 2.33 S.E.M., at pH 6.6 killing index = 52.3% ± 3.67 S.E.M.. Temperature was the only factor to significantly affect the killing indices among temperature and oxygen treatment groups. The killing index was lowest (29.3% ± 3.25 S.E.M.) at 30 °C and 7% oxygen, simulating in vivo oxygen pressure in well-aerated conditions and 30 °C and 3% oxygen, simulating in vivo oxygen pressure in hypoxia (30.5% ± 3.25 S.E.M.), compared with the index in 7% oxygen at low temperature (18 °C) (44.4% ± 4.50 S.E.M.) or compared with low oxygen (3%) at low temperature (18 °C) (39.7% ± 2.51 S.E.M.). The seasonal and diurnal rise in temperature may, therefore, be an important factor contributing to summer mortalities of C. gigas.     

Physiological bases of genetically determined variation in growth of marine invertebrate larvae: A study of growth heterosis in the bivalve Crassostrea gigas

Many species of marine animals have larval stages whose rates of growth in the plankton are regulated by complex combinations of biological and environmental factors. In this study, we focus on the physiological bases that underlie endogenous variation in growth potential of larvae. Our approach was based on experimental crosses of gravid adults from pedigreed families of the Pacific oyster, Crassostrea gigas. This produced large numbers of larvae with different growth rates when reared under similar environmental conditions of food and temperature. A total of 35 larval families were reared to test hypotheses regarding the physiological bases of growth variation. Growth rate of these larval families varied over a five-fold range, from 3.4 (± 0.5, S.E.) to 17.6 (± 0.6) μm day^− 1. The suite of integrated measurements applied to study growth variation included size, biochemical compositions, rates of particulate and dissolved nutrient acquisition, absorption efficiencies, respiration rates and enzyme activities. We show that a complex set of physiological processes regulated differences in genetically determined growth rates of larvae. One-half of the energy required for faster growth came from an enhanced, size-specific feeding ability. Differences in absorption rates were not significant for slow- and fast-growing larvae, nor were differences in size-specific respiration rates. Metabolic processes accounted for the additional 50% of the energy “savings” required to explain enhanced growth rates. We propose that different protein depositional efficiencies could account for this energy saving. Quantitative analyses of the endogenous physiological factors that cause variation in growth rate will allow for a more sophisticated understanding of growth, survival and recruitment potential of larvae.