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.     

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.     

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.     

Heterologous expression of the <Emphasis Type="Italic">Crassostrea gigas</Emphasis> (Pacific oyster) alternative oxidase in the yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Alternative oxidase (AOX) is a terminal oxidase within the inner mitochondrial membrane (IMM) present in many organisms where it functions in the electron transport system (ETS). AOX directly accepts electrons from ubiquinol and is therefore capable of bypassing ETS Complexes III and IV. The human genome does not contain a gene coding for AOX, so AOX expression has been suggested as a gene therapy for a range of human mitochondrial diseases caused by genetic mutations that render Complex III and/or IV dysfunctional. An effective means of screening mutations amenable to AOX treatment remains to be devised. We have generated such a tool by heterologously expressing AOX from the Pacific oyster (Crassostrea gigas) in the yeast Saccharomyces cerevisiae under the control of a galactose promoter. Our results show that this animal AOX is monomeric and is correctly targeted to yeast mitochondria. Moreover, when expressed in yeast, Pacific oyster AOX is a functional quinol oxidase, conferring cyanide-resistant growth and myxothiazol-resistant oxygen consumption to yeast cells and isolated mitochondria. This system represents a high-throughput screening tool for determining which Complex III and IV genetic mutations in yeast will be amenable to AOX gene therapy. As many human genes are orthologous to those found in yeast, our invention represents an efficient and cost-effective way to evaluate viable research avenues. In addition, this system provides the opportunity to learn more about the localization, structure, and regulation of AOXs from animals that are not easily reared or manipulated in the lab.     

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.     

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.     

<Emphasis Type="Italic">Crassostrea gigas</Emphasis> in natural oyster banks in southern Brazil

We report on the invasion of Brazil by the Pacific oyster Crassostrea gigas, and discuss the likely routes of invasion. Because this phenotypically diverse oyster sometimes resembles the native species C. brasiliana and C. rhizophorae, its invasion went unnoticed until it was detected through the analysis of DNA sequences for ribosomal 16S and the ribosomal second internal transcribed spacer. C. gigas was found amongst the native species in oyster banks up to 100 km south of oyster farms in South Brazil. Under most circumstances, water temperatures in the coastal southerly Brazil current would be too high to allow for the establishment of stable populations of C. gigas, but the production of spat in oyster farm laboratories has probably selected for resistance to warmer temperatures, which would promote invasion by C. gigas.     

Possible Probiotic Lactic Acid Bacteria Isolated from Oysters (<Emphasis Type="Italic">Crassostrea gigas</Emphasis>)

We attempted to isolate lactic acid bacteria (LAB) from the marine oyster (Crassostrea gigas) and selected several environmental stress-resistant isolates for the development of a future probiotic adjuvant for marine aquaculture. Twenty-six presumptive LAB isolates were extracted from oysters and screened (by an agar diffusion assay) for antimicrobial activity toward various pathogens: Vibrio parahaemolyticus, Streptococcus iniae, and Edwardsiella tarda. Eight isolates had an antibacterial activity toward V. parahaemolyticus; in particular, 6 isolates showed a growth-inhibitory activity, with inhibition zone diameters > 15 mm. Of these, 5 isolates (JL17, JL18, JL28, HL7, and HL32) were also active against S. iniae and E. tarda. Enterococcus faecium HL7 was selected as the isolate most resistant to environmental stressors: the minimum NaCl, ethanol, and hydrogen peroxide concentrations at which HL7 cells lost their viability were 1.9 M, 11%, and 0.013%, respectively. When an antibiotic sensitivity test was performed on E. faecium HL7, this isolate was found to be resistant to trimethoprim/sulfamethoxazole, cephalothin, ampicillin, rifampin, gentamicin, cefotaxime, cefepime, cefotetan, nalidixic acid, and kanamycin. While the oyster model studies provided indication that E. faecium HL7 could be a good candidate as biocontrol agent against V. vulnificus, further optimization is needed in the actual animal rearing situation.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

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.     

Expression and DNA methylation pattern of reproduction-related genes in partially fertile triploid Pacific oysters <Emphasis Type="Italic">Crassostrea gigas</Emphasis>

Partial or complete sterility is an obvious feature in triploid Pacific oyster (Crassostrea gigas) which contributes to improving rearing performances. Despite the significance of sterility, the molecular mechanism behind it remains elusive and related research was limited. This study focused on six reproduction-related genes and compared their different behavior in gene expression and DNA methylation pattern between triploid and diploid oysters in order to provide more molecular information. The gonadal development of triploid oyster was examined by histology before molecular analysis. Gametogenesis disturbance was observed in triploid oysters at different development stages (stage II and III) with more serious impairment in females. QPCR showed significant gene expression difference between diploid and triploid in two genes: putative Vg and cgER. Gene expression of putative Vg was delayed in triploids while for cgER triploid oyster showed higher expression and the difference was significant at stage III. DNA methylation pattern of these two genes were further investigated by bisulfite sequencing. Between diploid and triploid oysters, no difference was observed in total methylation level but some individual loci showed different patterns: significantly high methylation rate of loci 2284 in cgER was observed in triploid oyster which has a higher expression of this gene. This study indicated that putative Vg and cgER might play a role in partial sterile in triploid C. gigas. Gene expression could be regulated by the methylation pattern at specific individual locus, which deserves equivalent attention as well as total DNA methylation level.