AFLP-Based Genetic Linkage Maps of the Pacific Oyster <Emphasis Type="Italic">Crassostrea gigas</Emphasis> Thunberg

Amplified fragment length polymorphisms (AFLPs) were used for genome mapping in the Pacific oyster Crassostrea gigas Thunberg. Seventeen selected primer combinations produced 1106 peaks, of which 384 (34.7%) were polymorphic in a backcross family. Among the polymorphic markers, 349 were segregating through either the female or the male parent. Chi-square analysis indicated that 255 (73.1%) of the markers segregated in a Mendelian ratio, and 94 (26.9%) showed significant (P < 0.05) segregation distortion. Separate genetic linkage maps were constructed for the female and male parents. The female framework map consisted of 119 markers in 11 linkage groups, spanning 1030.7 cM, with an average interval of 9.5 cM per marker. The male map contained 96 markers in 10 linkage groups, covering 758.4 cM, with 8.8 cM per marker. The estimated genome length of the Pacific oyster was 1258 cM for the female and 933 cM for the male, and the observed coverage was 82.0% for the female map and 81.3% for the male map. Most distorted markers were deficient for homozygotes and closely linked to each other on the genetic map, suggesting the presence of major recessive deleterious genes in the Pacific oyster.     

A Functional Study of Transforming Growth Factor-Beta from the Gonad of Pacific Oyster <Emphasis Type="Italic">Crassostrea gigas</Emphasis>

The transforming growth factor (TGF)-β superfamily is a group of important growth factors involved in multiple processes such as differentiation, cell proliferation, apoptosis and cellular growth. In the Pacific oyster Crassostrea gigas, the oyster gonadal (og) TGF-β gene was recently characterized through genome-wide expression profiling of oyster lines selected to be resistant or susceptible to summer mortality. Og TGF-β appeared specifically expressed in the gonad to reach a maximum when gonads are fully mature, which singularly contrasts with the pleiotropic roles commonly ascribed to most TGF-β family members. The function of og TGF-β protein in oysters is unknown, and defining its role remains challenging. In this study, we develop a rapid bacterial production system to obtain recombinant og TGF-β protein, and we demonstrate that og TGF-β is processed by furin to a mature form of the protein. This mature form can be detected in vivo in the gonad. Functional inhibition of mature og TGF-β in the gonad was conducted by inactivation of the protein using injection of antibodies. We show that inhibition of og TGF-β function tends to reduce gonadic area. We conclude that mature og TGF-β probably functions as an activator of germ cells development in oyster.     

Evidence for the Involvement of Pathogenic Bacteria in Summer Mortalities of the Pacific Oyster <Emphasis Type="Italic">Crassostrea gigas</Emphasis>

A study was conducted to investigate the involvement of bacteria in oyster mortalities during summer. Moribund and apparently healthy oysters were sampled during mortality events along the French coast and in rearing facilities, usually when temperature reached 19°C or higher, and oysters were in the gonadal maturation phase. Hemolymph samples were aseptically withdrawn and submitted to bacteriological analysis. In healthy oysters, bacteria colonized hemolymph at low concentrations depending on the location. In most moribund oysters, bacteria were present in hemolymph and other tissues. These bacterial populations were more often diverse in oysters originating from the open sea than from facilities where animals were generally infected by a single type of bacterium. Only the dominant colonies were identified by phenotypic and genotypic characters (RFLP of GyrB gene and partial sequence of 16S rRNA gene). They belonged to a limited number of species including Vibrio aestuarianus, members of the V. splendidus group, V. natriegens, V. parahaemolyticus, and Pseudoalteromonas sp. The most frequently encountered species was V. aestuarianus (56% of isolates), which was composed of several strains closely related by their 16S rRNA gene but diverse by their phenotypic characters. They appeared intimately linked to oysters. The species within the V. splendidus group were less prevalent (25% of isolates) and more taxonomically dispersed. A majority of the dominant strains of V. aestuarianus and V. splendidus group injected to oysters induced mortality, whereas others belonging to the same species, particularly those found in mixture, appeared innocuous.     

Relationship Among Intron Length,Gene Expression,and Nucleotide Diversity in the Pacific Oyster <Emphasis Type="Italic">Crassostrea gigas</Emphasis>

Crassostrea gigas is a model mollusk, but its genetic features have not been studied comprehensively. In this study, we used whole-genome resequencing data to identify and characterize nucleotide diversity and population recombination rate in a diverse collection of 21 C. gigas samples. Our analyses revealed that C. gigas harbors both extremely high genetic diversity and recombination rates across the whole genome as compared with those of the other taxa. The noncoding regions, introns, intergenic spacers, and untranslated regions (UTRs) showed a lower level diversity than the synonymous sites. The larger introns tended to have lower diversity. Moreover, we found a negative association of the non-synonymous diversity with gene expression, which suggested that purifying selection played an important role in shaping genetic diversity. The nucleotide diversity at the 100- and 50-kb levels was positively correlated with population recombination rates, which was expected if the diversity was shaped by purifying selection or hitchhiking of advantageous mutants. Our work gives a general picture of the oyster’s polymorphism pattern and its association with recombination rates.     

Chromosomal organization of simple sequence repeats in the Pacific oyster (<Emphasis Type="Italic">Crassostrea gigas</Emphasis>): (GGAT)<Subscript>4</Subscript>, (GT)<Subscript>7</Subscript> and (TA)<Subscript>10</Subscript> chromosome patterns

Chromosome identification is essential in oyster genomic research. Fluorescence in situ hybridization (FISH) offers new opportunities for the identification of oyster chromosomes. It has been used to locate satellite DNAs, telomeres or ribosomal DNA sequences. However, regarding chromosome identification, no study has been conducted with simple sequence repeats (SSRs). FISH was used to probe the physical organization of three particular SSRs, (GGAT)(4), (GT)(7) and (TA)(10) onto metaphase chromosomes of the Pacific oyster, Crassostrea gigas. Hybridization signals were observed in all the SSR probes, but the distribution and intensity of signals varied according to the oligonucleotide repeat. The intercalary, centromeric and telomeric bands were observed along the chromosomes, and for each particular repeat every chromosome pair presented a similar pattern, allowing karyotypic analysis with all the SSRs tested. Our study is the first in mollusks to show the application of SSR in situ hybridization for chromosome identification and karyotyping. This technique can be a useful tool for oyster comparative studies and to understand genome organization in different oyster taxa.     

Elevated CO<Subscript>2</Subscript> influences herbivory-induced defense responses of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

We experimentally demonstrate that elevated CO₂ can modify herbivory-induced plant chemical responses in terms of both total and individual glucosinolate concentrations. Overall, herbivory by larvae of diamondback moths (Plutella xylostella) resulted in no change in glucosinolate levels of the annual plant Arabidopsis thaliana under ambient CO₂ conditions. However, herbivory induced a significant 28–62% increase in glucosinolate contents at elevated CO₂. These inducible chemical responses were both genotype-specific and dependent on the individual glucosinolate considered. Elevated CO₂ can also affect structural defenses such as trichomes and insect-glucosinolate interactions. Insect performance was significantly influenced by specific glucosinolates, although only under CO₂ enrichment. This study can have implications for the evolution of inducible defenses and coevolutionary adaptations between plants and their associated herbivores in future changing environments.     

Elevated CO<Subscript>2</Subscript> ameliorated oxidative stress induced by elevated O<Subscript>3</Subscript> in <Emphasis Type="Italic">Quercus mongolica</Emphasis>

Using open top chambers, the effects of elevated O₃ (80 nmol mol⁻¹) and elevated CO₂ (700 μmol mol⁻¹), alone and in combination, were studied on young trees of Quercus mongolica. The results showed that elevated O₃ increased malondialdehyde content and decreased photosynthetic rate after 45 days of exposure, and prolonged exposure (105 days) induced significant increase in electrolyte leakage and reduction of chlorophyll content. All these changes were alleviated by elevated CO₂, indicating that oxidative stress on cell membrane and photosynthesis was ameliorated. After 45 days of exposure, elevated O₃ stimulated activities of superoxide dismutase (SOD, EC 1.15.1.1) and ascorbate peroxidase (APX, EC 1.11.1.11), but the stimulation was dampened under elevated CO₂ exposure. Furthermore, ascorbate (AsA) and total phenolics contents were not higher in the combined gas treatment than those in elevated O₃ treatment. It indicates that the protective effect of elevated CO₂ against O₃ stress was achieved hardly by enhancing ROS scavenging ability after 45 days of exposure. After 105 days of exposure, elevated O₃ significantly decreased activities of SOD, catalase (CAT, EC 1.11.1.6) and APX and AsA content. Elevated CO₂ suppressed the O₃-induced decrease, which could ameliorate the oxidative stress in some extent. In addition, elevated CO₂ increased total phenolics content in the leaves both under ambient O₃ and elevated O₃ exposure, which might contribute to the protection against O₃-induced oxidative stress as well.     

Massive settlements of the Pacific oyster, <Emphasis Type="Italic">Crassostrea gigas</Emphasis>, in Scandinavia

The Pacific oyster (Crassostrea gigas) is an important aquaculture species world-wide. Due to its wide environmental tolerance and high growth rate, it has also become a successful invader in many areas, leading to major ecosystem changes. Low water temperatures were previously believed to restrict the establishment of Pacific oysters in Scandinavia. However, recent surveys reveal that the Pacific oyster is now established in many areas in Scandinavia. We present data on the current distribution, abundance and age-structure in Denmark, Sweden and Norway. The biomass of oysters in the Danish Wadden Sea increased from 1,056 to 6,264 tonnes between 2005 and 2007. Massive settlements were observed along the Swedish west coast in 2007, with densities >400 oysters per m⁻². In Norway, populations are established on the southern coast, and specimens have been found as far north as 60°N. The potential impacts and probable causes of this recent large-scale establishment are discussed.     

Elevated CO<Subscript>2</Subscript> influences host plant defense response in chickpea against <Emphasis Type="Italic">Helicoverpa armigera</Emphasis>

Global atmospheric concentration of CO₂ is likely to increase from 350 to 750 ppm over the next 100 years. The present studies were undertaken to understand the effects of elevated CO₂ on enzymatic activity and secondary metabolites in chickpea in relation to expression of resistance to pod borer, Helicoverpa armigera. Fifteen-day-old chickpea plants [ICCL 86111—resistant and JG 11—commercial cultivar] grown in the greenhouse were transferred to open-top chambers (OTC) and kept under 350, 550 and 750 ppm of CO₂. Twenty neonates of H. armigera were released on each plant at 7 days after shifting the pots to the OTCs. Un-infested plants were maintained as controls. After 7 days of infestation, the activities of defensive enzymes [peroxidase (POD), polyphenol oxidase (PPO), phenylalanine ammonia lyase (PAL) and tyrosine ammonia lyase (TAL)] and amounts of total phenols and condensed tannins increased with an increase in CO₂ concentration in chickpea. The nitrogen balance index was greater in plants kept at 350 ppm CO₂ than in plants kept under ambient conditions. The H. armigera-infested plants had higher H₂O₂ content; amounts of oxalic and malic acids were greater at 750 ppm CO₂ than at 350 ppm CO₂. Plant damage was greater at 350 ppm than at 550 and 750 ppm CO₂. This information will be useful for understanding effects of increased levels of CO₂ on expression of resistance to insect pests to develop strategies to mitigate the effects of climate change.     

Trojan Horse Strategy for Non-invasive Interference of <Emphasis Type="Italic">Clock</Emphasis> Gene in the Oyster <Emphasis Type="Italic">Crassostrea gigas</Emphasis>

RNA interference is a powerful method to inhibit specific gene expression. Recently, silencing target genes by feeding has been successfully carried out in nematodes, insects, and small aquatic organisms. A non-invasive feeding-based RNA interference is reported here for the first time in a mollusk bivalve, the pacific oyster Crassostrea gigas. In this Trojan horse strategy, the unicellular alga Heterocapsa triquetra is the food supply used as a vector to feed oysters with Escherichia coli strain HT115 engineered to express the double-stranded RNA targeting gene. To test the efficacy of the method, the Clock gene, a central gene of the circadian clock, was targeted for knockout. Results demonstrated specific and systemic efficiency of the Trojan horse strategy in reducing Clock mRNA abundance. Consequences of Clock disruption were observed in Clock-related genes (Bmal, Tim1, Per, Cry1, Cry2, Rev.-erb, and Ror) and triploid oysters were more sensitive than diploid to the interference. This non-invasive approach shows an involvement of the circadian clock in oyster bioaccumulation of toxins produced by the harmful alga Alexandrium minutum.     

Elevated CO<Subscript>2</Subscript> increases energy-use efficiency of invasive <Emphasis Type="Italic">Wedelia trilobata</Emphasis> over its indigenous congener

Increasing atmospheric CO₂ concentration is regarded as an important factor facilitating plants invasions by stimulating invasive species growth. However, the physiological mechanisms by which invasive plants increase at the expense of existing native plants are poorly understood. Plant growth is always related to energy-use process including energy assimilation and expenditure, and thus examination of energetic properties could provide mechanistic insight into growth responses to increased CO₂. The aims of this study were to examine the effect of rising CO₂ on the growth and energetic properties of alien invasive species (Wedelia trilobata (L.) Hitchc.) and its native congener (Wedelia chinensis (Osbeck.) Merr.) in South China, and to determine if the specific energetic properties of invasive species at elevated CO₂ favoring its growth. Elevated CO₂ stimulated a greater increase in biomass production for invasive W. trilobata (58.9%) than for its indigenous congener (48.1%). Meanwhile, elevated CO₂ altered the energetic properties differently upon species. For invasive W. trilobata, elevated CO₂ significantly increased total energetic gain via photosynthetic activity (A _total), but decreased energetic cost of biomass construction (CC), and thus enhanced photosynthetic energy-use efficiency (PEUE) by 85.3%. In contrast, the indigenous W. chinensis showed a slight increase in PEUE by 43.8%. Additionally, W. trilobata individuals grown in elevated CO₂ increased energy allocation towards stems. Statistic analysis revealed significant associations between growth characteristics (relative growth rate and biomass) and energetic properties (CC and PEUE), suggesting the greater growth stimulation in invasive species could be partly explained by its specific energetic properties in elevated CO₂ concentration. The invasive species showed a greater increase in energy-use efficiency under elevated CO₂, which consequently facilitated its growth. It might be a physiological mechanism promoting success of invasion with ongoing increase in atmospheric CO₂ concentration.     

Physiological responses of <Emphasis Type="Italic">Abies faxoniana</Emphasis> populations from different elevations to increased CO<Subscript>2</Subscript> and N application

The altitude-related responses to the increased application of CO₂, N, and their combination were investigated in two Abies faxoniana populations, which originated from a subalpine coniferous forest at elevations of 2,580 and 3,200 m using closed-top chambers. The two contrasting populations were subjected to two CO₂ regimes (350 and 700 µmol mol^?1) and two N levels (0 and 5 g N m^?2 year^?1). Their net photosynthetic rate, non-structural carbohydrate concentration, and photosynthetic N use efficiency (PNUE) increased under elevated CO₂. However, the increases detected in the high-elevation (HE) population were significantly greater than those found in the low-elevation (LE) population. Under elevated CO₂ and N application, the maximal carboxylation rate (V _cmax) increased in HE population, whereas no effects were found on V _cmax in LE population. The C to N ratio decreased under N application in both populations. N application also induced the HE population to show greater increases in free amino acids, soluble proteins, N concentration, and PNUE than LE population. These results suggested that the population from HE was more sensitive to elevated CO₂ and (or) N application than LE population. Results of this study provided valuable knowledge for predicting forest development under increased atmospheric CO₂ concentration and (or) N deposition.     

Responses of tropical native and invader C<Subscript>4</Subscript> grasses to water stress,clipping and increased atmospheric CO<Subscript>2</Subscript> concentration

The invasion of African grasses into Neotropical savannas has altered savanna composition, structure and function. The projected increase in atmospheric CO₂ concentration has the potential to further alter the competitive relationship between native and invader grasses. The objective of this study was to quantify the responses of two populations of a widespread native C₄ grass (Trachypogon plumosus) and two African C₄ grass invaders (Hyparrhenia rufa and Melinis minutiflora) to high CO₂ concentration interacting with two primary savanna stressors: drought and herbivory. Elevated CO₂ increased the competitive potential of invader grasses in several ways. Germination and seedling size was promoted in introduced grasses. Under high CO₂, the relative growth rate of young introduced grasses was twice that of native grass (0.58 g g⁻¹ week⁻¹ vs 0.25 g g⁻¹ week⁻¹). This initial growth advantage was maintained throughout the course of the study. Well-watered and unstressed African grasses also responded more to high CO₂ than did the native grass (biomass increases of 21–47% compared with decreases of 13–51%). Observed higher water and nitrogen use efficiency of invader grasses may aid their establishment and competitive strength in unfertile sites, specially if the climate becomes drier. In addition, high CO₂ promoted lower leaf N content more in the invader grasses. The more intensive land use, predicted to occur in this region, may interact with high CO₂ to fincreasesavor the African grasses, as they generally recovered faster after simulated herbivory. The superiority of invader grasses under high CO₂ suggests further in their competitive strength and a potential increased rate of displacement of the native savannas in the future by grasslands dominated by introduced African species.     

Estimates of Heritability for Growth and Shell Color Traits and Their Genetic Correlations in the Black Shell Strain of Pacific Oyster <Emphasis Type="Italic">Crassostrea gigas</Emphasis>

The Pacific oyster Crassostrea gigas has been introduced widely and massively and became an economically important aquaculture species on a global scale. We estimated heritabilities of growth and shell color traits and their genetic correlations in black shell strain of C. gigas. Analyses were performed on 22 full-sib families in a nested mating design including 410 individuals at harvest (24 months of age). The parentage assignment was inferred based on four panels of multiplex PCR markers including 10 microsatellite loci and 94.9% of the offspring were unambiguously assigned to single parent pairs. The Spearman correlation test (r = ? 0.992, P < 0.001) demonstrated the high consistency of the shell pigmentation (SP) and L* and their same efficacy in shell color measurements. The narrow-sense heritability estimated under the animal model analysis was 0.18 ± 0.12 for shell height, 0.25 ± 0.16 for shell length, 0.10 ± 0.09 for shell width, 0.42 ± 0.20 for total weight, 0.32 ± 0.18 for shell weight, and 0.68 ± 0.16 for L*, 0.69 ± 0.16 for shell pigmentation, respectively. The considerable additive genetic variation in growth and shell color traits will make it feasible to produce genetic improvements for these traits in selective breeding program. High genetic and phenotypic correlations were found among growth traits and among shell color traits. To optimize a selection strategy for both fast growth and pure dark shell strain of C. gigas, it is proposed to take both total weight and black shell as joint objective traits in selective breeding program. Our study offers an important reference in the process of selective breeding in black shell color stain of C. gigas and will facilitate to develop favorable breeding strategies of genetic improvements for this economically important strain.