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.     

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.     

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.     

Characterization of the quantitatively major collagen in the mantle of oyster <Emphasis Type="Italic">Crassostrea gigas</Emphasis>

ABSTRACT:   A quantitatively major collagen was isolated from the pepsin-solubilized collagen preparation of the mantle by differential salt precipitation and phosphocellulose column chromatography, and its constituent α components (α1 and α2) were purified by phosphocellulose column chromatography. The subunits were demonstrated to be genetically distinct from each other by peptide mapping and amino acid analysis. The amino acid composition calculated from those of the α1 and α2 components in 2 : 1 ratio coincided well with that of the major collagen from the mantle. These results suggest that the major collagen in the mantle of the oyster may have a heterotrimer structure (α1)₂α2.     

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.     

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.     

Genetic variation and population structure of the Pacific cod <Emphasis Type="Italic">Gadus macrocephalus</Emphasis> in Korean waters revealed by mtDNA and msDNA markers

To investigate the extent of genetic differentiation among wild populations of the Pacific cod Gadus macrocephalus, we have examined genetic polymorphism at five locations within Korean waters [Boryeong in the West Sea (WC-BR); Jinhae Bay in the South Sea (SC-JH); Jumunjin (EC-JM), Jukbyeon (EC-JB), and Bangeojin (EC-BJ) off the eastern coast of Korea] using mitochondrial DNA (mtDNA) control region and microsatellite DNA (msDNA) markers. Nucleotide sequence analysis of 584 bp in the variable portion of the 5′ end of the mtDNA control region revealed 27 variable nucleotide sites among 184 individuals, which defined eight, three, and 11 haplotypes in the western, southern, and eastern coast populations, respectively. The mtDNA analysis revealed a low variability but significant local differentiation among populations from these three areas within Korean waters. msDNA analysis also revealed moderate polymorphism in the wild populations, with a mean of 13.8–22.6 alleles per locus for the five msDNA markers and observed (and expected) heterozygosities of 0.755 (0.825) for the WC-BR, 0.793 (0.810) for the SC-JH, 0.920 (0.905) for the EC-BJ, 0.783 (0.865) for the EC-JB, and 0.804 (0.812) for the EC-JM populations. Analysis of msDNA loci indicated that Pacific cod sampled at the WC-BR, SC-JH, and EC-JB sites belong to genetically distinct populations. However, no significant difference was found between the Pacific cod population from SC-JH and that from EC-BJ. Consequently, three genetically distinct populations, namely, WC-BR, SC-JH and EC-BJ, and EC-JB, were identified using msDNA analysis. These results indicate that genetically distinct populations of Pacific cod are present in Korean coastal waters where spawning aggregations occur.     

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.     

Growth and survival of grouper <Emphasis Type="Italic">Epinephelus</Emphasis><Emphasis Type="Italic">coioides</Emphasis> (Hamilton) larvae fed free-living nematode <Emphasis Type="Italic">Panagrellus redivivus</Emphasis> at first feeding

The free-living nematode, Panagrellus redivivus, was tested as live food for grouper Epinephelus coioides larvae during the first feeding stage. A series of experiments were conducted to determine the acceptability of the free-living nematodes in grouper larvae at first feeding, the optimum nematode density and the response of the larvae to nutritionally enriched nematode. All experiments were conducted in 200-L conical tanks filled with 150-L filtered seawater and stocked at 15 larvae L⁻¹. Duration of feeding experiments was up to day 21 (experiment 1) and 14 days (experiment 2 and 3). Brachionus plicatilis and Artemia (experiment 1) and Brachionus plicatilis alone (experiment 2 & 3) was used as the control treatment. Observations indicated that the grouper larvae readily fed on free-living nematodes as early as 3 days posthatching, the start of exogenous feeding. Optimum feeding density for the larvae was 75 nematodes ml⁻¹. The enrichment of cod liver oil or sunflower oil influenced the total lipids and n-3 highly unsaturated fatty acids of P. redivivus, which in turn influenced those of the grouper larvae, however, growth and survival of the larvae were not affected (P > 0.05). The results from this investigation showed that the nematode, P. redivivus, can be used as first live food for grouper larvae from the onset of exogenous feeding until they could feed on Artemia nauplii.     

Potential of cinnamon (<Emphasis Type="Italic">Cinnamomum verum</Emphasis>) oil to control <Emphasis Type="Italic">Streptococcus iniae</Emphasis> infection in tilapia (<Emphasis Type="Italic">Oreochromis niloticus</Emphasis>)

In this study, four essential oils—cinnamon oil, leech lime oil, lemongrass oil, and turmeric oil—were examined for their antimicrobial activities against Streptococcus iniae, a bacterium that is pathogenic in fish, in which it causes streptococcosis. Cinnamon oil was the most potent antimicrobial agent among these oils, with a minimal inhibitory concentration (MIC) of 40 μg/ml. By using gas chromatography–mass spectrometry (GC–MS), it was found that the major components of cinnamon oil were cinnamaldehyde (90.24), limonene (2.42%), cinnamyl acetate (2.03%), linalool (1.16%), and α-terpineol (0.87%). Of these compounds, only cinnamaldehyde exhibited antimicrobial activity against S. iniae, with an MIC of 20 μg/ml. In an in vivo trial, no mortality was apparent in fish fed on fish diets supplemented with 0.4% (w/w) of cinnamon oil and with 0.1% (w/w) of oxytetracycline 5 days prior to infection with S. iniae. These results indicate that cinnamon oil had a protective effect on experimental S. iniae infection in tilapia, and thus has the potential to replace the antibiotics used to control this disease.     

Prey fish selection by Far Eastern catfish <Emphasis Type="Italic">Silurus asotus</Emphasis> and largemouth bass <Emphasis Type="Italic">Micropterus salmoides</Emphasis>

ABSTRACT:   Prey fish selection by Far Eastern catfish and largemouth bass were examined using bluegill, Japanese dace and crucian carp as food fish. In both aquaria (1.2 m × 0.45 m) and ponds (2.8 m × 1.2 m), bluegill was not preyed on by catfish and bass more than dace and crucian carp. In aquaria, there was no significant difference in the consumption of dace and crucian carp between catfish and bass, but in ponds, catfish preyed on dace and crucian carp more and less than bass, respectively. In the case that only bluegill and catfish were introduced in ponds, catfish consumed 4–15 g of bluegill per day. The introduction of catfish into ponds and lakes for the purpose of eradicating bluegill is considered appropriate for areas with few native fish species.     

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.     

Recent expansion of Northeast Atlantic and Mediterranean populations of <Emphasis Type="Italic">Melicertus</Emphasis> (<Emphasis Type="Italic">Penaeus</Emphasis>) <Emphasis Type="Italic">kerathurus</Emphasis> (Crustacea: Decapoda)

We analysed the genetic diversity of Melicertus kerathurus (Penaeidae), a commercially valuable penaeid shrimp that is distributed in the Mediterranean Sea and eastern Atlantic Ocean. We examined the polymorphism of a 494 bp DNA segment of the mitochondrial COI region in 173 individuals, sampled in nine Mediterranean and two Atlantic samples, covering the whole range of the species from the tropical waters of the Gulf of Guinea to the eastern part of the Mediterranean Sea. The mean nucleotide and haplotype diversities were π = 0.00275 and h = 0.718, respectively, for the global data set, with the highest values occurring in the African samples and the lowest in the Adriatic Sea. A clear sample differentiation was found (F _st = 0.194), but this did not reflect a geographical pattern and there were only faint traces of an Atlantic–Mediterranean subdivision. Mismatch analysis and a high significant negative value of Tajima’s D suggested that M. kerathurus is not at mutation drift-equilibrium, but underwent a recent expansion after a period of low effective sample size. A postglacial recolonisation of the Mediterranean from an Atlantic refuge could be hypothesised based on these data.