Effect of starvation on biochemical composition and gametogenesis in the Pacific oyster <Emphasis Type="Italic">Crassostrea gigas</Emphasis>

The effects of starvation on biochemical composition and gametogenesis were investigated in the Pacific oyster Crassostrea gigas. Histological analysis, combined with oocyte examination and measurements of protein, glycogen and lipid levels and RNA/DNA ratio from gonad, adductor muscle and mantle tissue of each sex were performed. In the starved groups, C. gigas showed gonad development, but the progress was delayed during the experiment. Glycogen was the first substrate used by C. gigas for dealing with lack of food. While glycogen was rapidly consumed, protein and lipid contents decreased gradually. A decrease in the RNA/DNA ratio in the starved groups in all the body components was found during starvation, illustrating that RNA/DNA ratio was a valid indicator of nutritional condition in C. gigas. A significant increase in water and ash contents and a corresponding decrease in condition index were observed in the starved groups, showing that the water and ash content and condition index were related to the usage of glycogen, lipid and protein reserves in body composition. During starvation, energy reserves were mobilized for survival and gonad development, but spawning was arrested. The information obtained in this study is useful for broodstock management in the Pacific oyster industry.     

Selection response and realized heritability for growth in three stocks of the Pacific oyster <Emphasis Type="Italic">Crassostrea gigas</Emphasis>

To estimate response to selection and realized heritability for shell height, a one-generation selection was performed in the Pacific oyster Crassostrea gigas using three stocks from China (stock C), Japan (stock J), and Korea (stock K). Applying about the same intensity of selection in the upward direction, three selected and three control lines were created, which were reared under the same environmental conditions at larvae, spat, and grow-out stages. Stock C and stock J showed significantly higher response to selection and realized heritability than stock K at spat and grow-out stages (P < 0.05). At harvest on day 360, the selected lines of stocks C, J, and K were 12.2, 12.2, and 7.9% larger than their control lines, respectively, for shell height. When averaged across the grow-out period, the genetic gain for stocks C, J, and K was 13.2 ± 1.2, 13.2 ± 1.0, and 7.2 ± 0.7%, respectively, and realized heritability was 0.334 ± 0.028, 0.402 ± 0.024 and 0.149 ± 0.027, respectively. The relatively high realized heritability estimate obtained for stock C and stock J indicates that there is genetic variation in the two stocks and that selective breeding by mass selection is very promising.     

DNA methylation frequency and epigenetic variability of the Pacific oyster <Emphasis Type="Italic">Crassostrea gigas</Emphasis> in relation to the gametogenesis

The origin and maintenance of sexual gametogenesis might provide insights into eukaryote evolution. Epigenetic variation of eukaryote well-being would provide clues that could relate ecological and evolutionary processes. To investigate the relationship between gametogenic variability and epigenetic polymorphism in the Pacific oyster Crassostrea gigas, DNA methylation differences were represented by analyzing genome-wide methylation patterns using the fluorescent-labeled methylation sensitive-amplified polymorphism (F-MSAP) technique. The C. gigas genome was widely methylated and marked variations in the pattern of methylation were shown during female and male gonad development. Statistically, the total methylation level increased from early developing stage to resorbing stage, from 28.86 to 34.53% in ovaries, while it increased from 31.20 to 35.46% in this period in testes. The unweighted pair group method with arithmetic mean and principal coordinate analysis separated the C. gigas during female and male gonad development remarkably well, indicating remarkable differences across gametogenesis between ovaries and testes. Variation was higher in testes than in ovaries by the frequency based and multivariate analyses above. Both multivariate analyses and a significantly different population epigenetic structure suggested that a striking difference might exist across gametogenesis, reflecting a turning point of the methylation mechanism during reproductive progress. This study provides some information on the role of DNA methylation in the potential mechanism during female and male gonad development.     

Development and distribution of the non-indigenous Pacific oyster (<Emphasis Type="Italic">Crassostrea gigas</Emphasis>) in the Dutch Wadden Sea

Pacific oysters (Crassostrea gigas) were first observed in the Dutch Wadden Sea near Texel in 1983. The population increased slowly in the beginning but grew exponentially from the mid-1990s onwards, although now some stabilisation seems to be occurring. They occur on a variety of substrates such as mussel beds (Mytilus edulis), shell banks, dikes and poles. After initial settlement spat may fall on older individuals and congregate to dense clumps and subsequently form reefs. Individual Pacific oysters grow 3–4 cm long in their first year and 2–3 cm in their second year. Many mussel beds (Mytilus edulis) are slowly taken over by Pacific oysters, but there are also several reports of mussel spat settling on Pacific oyster reefs. This might in the end result in combined reefs. Successful Pacific oyster spat fall seems to be related to high summer temperatures, but also after mild summers much spat can be found on old (Pacific oyster) shells. Predation is of limited importance. Mortality factors are unknown, but every now and then unexplained mass mortality occurs. The gradual spread of the Pacific oyster in the Dutch Wadden Sea is documented in the first instance based on historical and anecdotal information. At the start of the more in-depth investigation in 2002, Pacific oysters of all size classes were already present near Texel. Near Ameland the development could be followed from the first observed settlement. On dense reefs each square metre may contain more than 500 adult Pacific oysters, weighing more than 100 kg per m2 fresh weight.     

Characterization of <Emphasis Type="Italic">kiss2</Emphasis> and <Emphasis Type="Italic">kissr2</Emphasis> genes and the regulation of kisspeptin on the HPG axis in <Emphasis Type="Italic">Cynoglossus semilaevis</Emphasis>

Reproduction allows organisms to produce offspring. Animals shift from immature juveniles into mature adults and become capable of sexual reproduction during puberty, which culminates in the first spermiation and sperm hydration or ovulation. Reproduction is closely related to the precise control of the hypothalamic–pituitary–gonadal (HPG) axis. Kisspeptin peptides are considered as the important regulator of HPG axis in mammalian. However, the current understanding of kisspeptin in flatfish is not comprehensive. In this study, we cloned and analyzed the kiss2 and kissr2 genes in Cynoglossus semilaevis. Interesting alternative splicing in the 5′-untranslated regions (UTR) of the Cskissr2 gene was found. The expression profiles of Cskiss2 and Cskissr2 showed relative high messenger RNA (mRNA) levels at the late gastrula stage during embryonic development, at total length = 40 mm during early gonadal differentiation, and in the brains and gonads of all investigated tissues. These results suggested that the kisspeptin system participated in embryogenesis and in the regulation of gonadal differentiation and development. Considering that the control and regulatory mechanisms of kisspeptin in the central reproductive axis are still unclear, we documented that the intramuscular injection of kisspeptin caused different sGnRH and cGnRH mRNA levels in a dose- and tissue-dependent manner. The mRNA expressions of FSH and LH were stimulated in the ovary and were inhibited in the testis under the kisspeptin treatments. These results provided foundations for understanding the roles of kisspeptin in the neuroendocrine system in fish. The manipulation of the kisspeptin system may provide new opportunities to control the gonadal development and even reproduction in fish.     

Trials on Pacific oyster (<Emphasis Type="Italic">Crassostrea gigas</Emphasis> Thunberg) rearing in the middle Adriatic Sea by means of different trays

The aim of this study was to compare the growth of juvenile Pacific oysters (mean weight 2.22 g; mean length 26.8 mm) reared with the suspended technique using three different types of trays fixed to long lines at a mussel farm located in the middle Adriatic Sea. Growth, mortality rate and quality traits of oysters cultured in a new type of tray (EXP) were compared to those of specimens reared in conventional trays (CON) and in “poches” (POC). Culture density was fixed at 0.03 oysters cm⁻² in all the tray types. After 11 months of growth, oysters reared in all the tray types reached marketable size, weighing between 94.08 and 110 g without significant differences. Length (98.8–103.3 mm), width (61.1–70.5 mm) and height (31.6–34.1 mm) did not show significant variations. The condition index showed differences among EXP (93.17%) compared to CON (81%) and POC oysters (72.87%), and significant differences were found between those reared at the surface and the bottom, regardless of tray type. Concerning mortality, the EXP group (7.5%) recorded significantly smaller losses than CON (18.4%) trays and POC (31.6%).     

Microzooplankton as a food source for the Pacific oyster <Emphasis Type="Italic">Crassostrea gigas</Emphasis>: seasonal variation in gut contents and food availability

In this study I investigated the seasonal occurrence of microzooplankton in the gut of the Pacific oyster Crassostrea gigas. Gut contents consisted of various microzooplankton, including tintinnid ciliates, dinoflagellates (Dinophysis spp.), copepods, copepod nauplii, bivalve larvae, and rotifers. Monthly variations in the abundance of protists (tintinnids and Dinophysis spp.) in the gut contents were significantly correlated with the variation in their abundances in the surrounding seawater, whereas this relationship was not significant with metazoans (copepods, copepod nauplii, and bivalve larvae). In laboratory experiments, oysters actively fed on ciliates. When oysters fed on ciliates labeled with a stable nitrogen isotope (¹⁵N), the ¹⁵N:total N ratio of the oyster tissues was significantly higher than that for oysters under a no-food condition, suggesting that oysters assimilate nitrogen of the ciliates into their body tissues. Based on these results, I conclude that microzooplankton forms an important food source for oysters, especially when other food sources such as phytoplankton are not abundant.     

Spatial learning-induced <Emphasis Type="Italic">egr</Emphasis>-<Emphasis Type="Italic">1</Emphasis> expression in telencephalon of gold fish <Emphasis Type="Italic">Carassius auratus</Emphasis>

The immediate-early gene (egr-1) expression was used to examine the neuron’s response in telencephalon of goldfish during spatial learning in small space. Fishes were pre-exposed in the experimental apparatus and trained to pick food from the tray in a rectangular-shaped arena. The apparatus was divided into identical compartments comprising three gates to provide different spatial tasks. After the fish learned to pass through the gate one, two more gates were introduced one by one. Fish made more number of attempts and took longer time (P < 0.05) to pass through the first gate than the gate two or three. This active learning induces the expression of egr-1 in telencephalon as established by western blot analysis. Subsequently, the fish learn quickly to cross the similar type of second and third gate and make fewer errors with a corresponding decline in the level of egr-1 expression. As the fish learned to pass through all the three gates, third gate was replaced by modified gate three. Interestingly, the level of egr-1 expression increased again, when the fish exhibit a high exploratory behavior to cross the modified gate three. The present study shows that egr-1 expression is induced in the telencephalon of goldfish while intensively acquiring geometric spatial information to pass through the gates.     

Evaluation of three phytoplankton species as food for the pearl oyster <Emphasis Type="Italic">Pinctada fucata</Emphasis>

In the pearl cultivation farms of the Ehime Prefecture, Japan, mass mortalities of the pearl oyster Pinctada fucata have occurred since 1994. The occurrences of mass mortality roughly coincided with a shift of the dominant phytoplankton from Skeletonema and Chaetoceros to Chaetoceros and Nitzschia all of which belong to Bacillariophyceae. Hence, we evaluated Nitzschia, together with Chaetoceros and Isocrysis, as food for the oyster. Wet weights, lengths, widths, glycogen contents, and growth rates in terms of wet weight of the oysters in all the feeding treatments were significantly higher than those in the non-feeding treatment. The highest glycogen content (2.34%) and growth rate (2.21 g month⁻¹) were found in the Chaetoceros treatment. Growth rate in the Isocrysis treatment (1.63 g month⁻¹) was also high, although glycogen content in this treatment (0.41%) was low. In the Nitzschia treatment, growth rate of the oyster (0.94 g month⁻¹) was the lowest and glycogen content (0.83%) was also low relative to that in the Chaetoceros treatment. Chlorophyll a concentration in fecal pellets was lowest in the Nitzschia treatment (<2.7 μg mg⁻¹), suggesting more complete digestion of Nitzschia by the oyster. Thus, Nitzschia was edible and digestible but not assimilated by P. fucata. We propose the following scenario for the relationship between Nitzschia dominance and mass mortality. When Nitzschia dominates in a culture area, the physiological condition of P. fucata deteriorates due to low assimilation of Nitzschia by the oyster, followed by susceptibility of the oyster to infection by agents lethal to the oyster.     

Molecular species diagnosis confirmed the occurrence of Kumamoto oyster <Emphasis Type="Italic">Crassostrea sikamea</Emphasis> in Korean waters

In order to develop an appropriate natural resource management policy, it is important to elucidate the cryptic habitats of species. Oyster species are likely to have such unrecognized habitats owing to the difficulty involved in morphological species identification. In this study, we discovered a novel cryptic habitat of the Kumamoto oyster Crassostrea sikamea, a sibling species of the Pacific oyster C. gigas, in Korean waters. We collected 50 oyster samples from Suncheon Bay, located in the southern Korean Peninsula, among which 20 individuals of C. sikamea, 29 of C. gigas, and one C. sikamea/C. gigas hybrid were identified based on mitochondrial and nuclear DNA markers. There were no significant differences in shell morphology between the two species, which is consistent with how difficult it is to delineate them based on their shell features. Our identification of this novel C. sikamea habitat should help to improve the resource management of East Asian oysters.     

Taurine transporter from the giant Pacific oyster <Emphasis Type="Italic">Crassostrea gigas</Emphasis>: function and expression in response to hyper- and hypo-osmotic stress

ABSTRACT:   Taurine is the primary osmolyte in marine molluscs, whose cellular osmo-conforming process is vital for environmental adaptation because of a lack of osmotic homeostasis. Here, cDNA cloning and expression, and functional analyses of taurine transporter (TAUT) from the giant Pacific oyster are reported on. The deduced amino-acid sequence of oyster TAUT (oyTAUT) showed 47–51% identity to those of vertebrate TAUT, whereas identity among the vertebrates is 78–95%. Functional analysis of oyTAUT expressed in Xenopus oocytes revealed that oyTAUT has a lower affinity and specificity for taurine and a requirement for higher NaCl concentration, compared with vertebrate TAUT. Taken together with similar functional properties of TAUT from mussel, indicated by our previous study, it is possible that these functional features reflect the internal environment of the molluscs (i.e. higher taurine and NaCl concentrations). Oyster taurine transporter mRNA expression was induced by not only hyper-osmotic stress, similar to other TAUT, but also hypo-osmotic stress. It is speculated that the expression in response to hypo-osmotic stress was induced by a substantial decrease in tissue taurine content following the decrease in the internal osmolality.     

The impact of successive mass selection on population genetic structure in the Pacific oyster (<Emphasis Type="Italic">Crassostrea gigas</Emphasis>) revealed by microsatellite markers

To evaluate the impact of mass selection on genetic structure in artificially closed populations of the Pacific oyster Crassostrea gigas, we performed mass selection over six generations on two stocks from Japan and Korea and analyzed their temporal genetic variation and structure using 18 microsatellite makers, which were compared with the base populations of the two selected lines and one wild population from China. The average numbers of alleles (Na), mean observed heterozygosities (Ho), and expected heterozygosities (He) varied over generations in the two selected lines (selected lines of Japan, Na = 10.7–14.9, Ho = 0.757–0.846, He = 0.778–0.871; selected lines of Korea, Na = 9.4–17.3, Ho = 0.736–0.865, He = 0.744–0.854). There was no significant reduction in heterozygosity in the two selected lines. However, the average number of alleles per locus was significantly lower in the fifth and sixth generations of the two selected lines compared with that in the base population and wild population (P < 0.05), suggesting that the successive mass selection in closed populations may increase the sensibility of rare alleles to genetic drift. Equalizing the sex ratio of parents and reducing the selection intensity properly with the increase of selective generations is recommended to minimize the deleterious effect of genetic drift and bottleneck caused by successive mass selection. The information obtained in this study is useful for the design of appropriate management strategies for selective breeding of C. gigas.     

Monitoring of <Emphasis Type="Italic">Francisella noatunensis</Emphasis> subsp. <Emphasis Type="Italic">orientalis</Emphasis> in farmed Nile tilapia (<Emphasis Type="Italic">Oreochromis niloticus</Emphasis>) in Brazil

Francisella noatunensis orientalis is a bacterium that causes emerging bacteriosis in Nile tilapia (Oreochromis niloticus) in many parts of the world, including Brazil. It is a non-motile, Gram-negative, strictly aerobic, facultative intracellular coccobacillus. This species of bacteria is responsible for low to high mortality in fish farms, causing economic losses for fish farmers. This study aimed to detect the presence of F. noatunensis orientalis using qPCR (real-time polymerase chain reaction) and to describe lesions caused by the bacterium in O. niloticus in Brazilian aquaculture. For this purpose, 360 fish from six fish farms (30 per farm) were sampled at two time points (n = 180 per sampling). Necropsies and histopathology were performed for lesion observation, in addition to qPCR and sequencing for detection and identification of Francisella species. Environmental data were collected using a multiparameter sonde YSI EXO2. All measured limnological variables were within the optimum range for cultivation of Nile tilapia. The major lesions present were melanization of the skin, splenomegaly, granulomas, and inflammatory cell responses. The prevalence of francisellosis varied from 0 to 86.66% between time periods and fish farms analyzed, and an outbreak was observed during the second sampling period. This study describes the prevalence of francisellosis in O. niloticus and reports that the lesions found are not exclusively associated with this bacterial disease.     

Characterization and expression of <Emphasis Type="Italic">lin-28a</Emphasis> involved in <Emphasis Type="Italic">lin28/let-7</Emphasis>signal pathway during early development of <Emphasis Type="Italic">P. olivaceus</Emphasis>

Heterochronic lin-28 is a conserved RNA-binding protein that plays a key role in the timing of developmental events in organisms. As a crucial heterochronic gene, the protein controls developmental events of the second of four larval stages in Caenorhabditi elegans. Heterochronic let-7 miRNAs are often present in various species and highly conserved in sequence and biological function and are required for various biological processes. Previous studies showed that ten let-7 miRNAs were identified in the Japanese flounder (Paralichthys olivaceus) and that they were primarily expressed during metamorphosis. In this study, we clone and characterize the lin-28a gene from P. olivaceus and exhibit its dynamic expression pattern at different developmental stages and various adult tissues. The results show that the P. olivaceus lin-28a gene has high sequence similarity with other species and is highly expressed in the embryonic stage but weakly expressed in the larval stage. In addition, lin-28a overexpression causes cell proliferation and significantly promotes the levels of pre-let-7a and pre-let-7d while markedly depressing let-7a and let-7d expression in FEC (Flounder Embryonic Cell), which indicate that lin-28 possibly blocks the maturation of let-7 miRNAs. Additionally, lin-28a is identified as a target gene of let-7 miRNAs, and let-7 miRNAs directly regulate lin-28a expression by targeting its 3′ UTR. Taken together, lin-28a along with let-7 miRNA participates in a lin-28/let-7 axis pathway that regulates cell division and timing of embryonic and metamorphic events in P. olivaceus.