Prolonged Culture Period on Production Cost and Factor Input: A Case from the Pacific Oyster,Crassostrea gigas,Farming Industry in Yunlin County,Taiwan

The Yunlin coastal area is the largest oyster culture in Taiwan; however, the oyster farmers reported the negative impact of a prolonged oyster culture period and an increased operating cost in 2010. This study uses the translog cost function to consider the possibility of an oyster culture period extension to estimate oyster cost elasticity, own‐price elasticities, and cross‐price (substitution) elasticities, to evaluate whether the oyster farming industry in Yunlin County has economies of scale, and to assess the relationship with a substitution of inputs. We found that the Yunlin oyster culture has economies of scale, and that the oyster farmers can expand production scale to reduce costs. The own‐price elasticities of demand for inputs are less than 1, indicating fairly inelastic factor demands in oyster production. The oyster farming industry displays strong substitutability between the prolonged culture period and capital input, suggesting that the oyster farming industry is more responsive to a higher prolonged culture period cost, in terms of capital input.     

Importance of <Emphasis Type="Italic">Gammarus</Emphasis> in aquaculture

In recent years, there have been significant increases in the production and breeding of aquaculture species; for example, the gilt-head (sea) bream and seabass in salt water and trout, carp, and tilapia in fresh water., Amphipods in the genus Gammarus are used as a rich source of protein to feed various pet animals such as aquarium fish, lizards, and turtles, as well as in aquatic ecotoxicology and water quality assessment. Gammarus meal is used as a partial replacement (10–20%) of fish meal. However, the reproduction and rearing of Gammarus in intensive or semi-intensive conditions has not yet been carried out. Gammarus products (live, dried flakes, or powdered) transported to the markets are constituted from wild caught Gammarus. Due to the high price of these products, Gammarus aquaculture potentially has great economic benefits. Therefore, there is a need to focus on the importance of Gammarus in aquaculture. In the present review, the ecology and distribution of Gammarus, its importance in aquatic ecotoxicology and water quality assessment, the chemical analysis of Gammarus and factors affecting chemical composition of it, and the importance of Gammarus in fish nutrition are discussed. In addition, the economic importance and culture methods of Gammarus are also reviewed. This review will be beneficial for scientific investigators and fish-crustacean farmers.     

Molecular cloning and expression of the heat shock protein 70 gene in the Kumamoto oyster <Emphasis Type="Italic">Crassostrea sikamea</Emphasis>

Episodes of summer mortality of the Kumamoto oyster Crassostrea sikamea are a major problem for its cultivation. Expression of the heat shock protein 70 (HSP70) is induced by various environmental stresses, including heat. We cloned and sequenced hsp70 complementary DNA from C. sikamea to investigate the relationship between hsp70 expression and heat tolerance in this oyster. Quantitative real-time polymerase chain reaction was performed using gill tissue dissected from oysters before and after heat shock for 1 h. The results showed hsp70 expression was faster and greater in oysters cultured at 20–22 °C than at 10–12 °C, and survival was lower among oysters cultured at 20–22 °C than at 10–12 °C. Moreover, heat tolerance was investigated by a 1-h pre-heat treatment, followed by exposure to heat shock conditions 5 days later. Survival was higher and hsp70 expression was notably lower in oysters that received the pre-heat treatment compared with those that did not. We conclude that a pre-heat treatment of only 1 h may be useful for inducing heat tolerance in C. sikamea, and that a low level of hsp70 expression after heat shock is an important index in selecting for high heat tolerance in these oysters.     

Development of a simple and rapid monoclonal antibody-based flow through immunogold assay (FIA) for detection of <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis>

In this study, we developed a simple, low-cost, and rapid flow through immunogold assay (FIA) for detection of Aeromonas hydrophila in fish tissues and validated. The developed assay relied on colloidal gold conjugated highly specific monoclonal antibody (B8E3) against 20 kDa protein of A. hydrophila. The assay can be completed within 5 min including antigen (sample) preparation and exhibited no cross-reaction with other major aquatic pathogens such as Vibrio anguillarum, V. parahaemolyticus, V. harveyi, white spot syndrome virus (WSSV), and Aphanomyces invadans. A wider range of Aeromonas species were tested including A. caviae, A. dhakensis, A. sobria, A. veronii, A. culicicola, A. sharmana, and A. schubertii and no cross-reactivity could be observed. The developed technique FIA could detect 10³ CFU/ml of aeromonad cells. In addition, the FIA is as sensitive as the first-step polymerase chain reaction (PCR) and 1000 times sensitive than the immunodot blot method. The validation study revealed the accuracy of FIA by comparing with the standard first-step PCR. The proposed FIA is low-cost, easy-to-use, and compatible for field-level analysis of A. hydrophila in fish tissues. Our developed FIA could successfully detect A. hydrophila at early stage of the infection and could be easily availed by farmers, fish producers, non-technicians, and technicians in the aquaculture industry.     

Long-term fluctuations in mollusk populations before and after the appearance of the alien predator <Emphasis Type="Italic">Euspira fortunei</Emphasis> on the Tona coast,Miyagi Prefecture,northern Japan

The impact of Euspira fortunei as an alien predator on fluctuations in population densities of prey mollusks between 2001 and 2010 on the Tona coast, northern Japan, was investigated. This species increased dramatically from 2002 to 2004. In contrast, prey species such as Ruditapes philippinarum and Macoma incongrua decreased rapidly from 2001 to 2004, whereas Pillucina pisidium and Batillaria cumingii did not show a significant decrease during this period. Using a laboratory experiment, we were able to show that these decreases in the population densities of some species of mollusks but not other species were the result of species-selective predation by E. fortunei, as the experiment revealed that E. fortunei preferred to attack R. philippinarum and M. incongrua rather than P. pisidium, although the predator did expand its diet to include P. pisidium after it had consumed 16 of the 20 preferred prey available (i.e., R. philippinarum and M. incongrua). This case study suggests that invasive naticid predators have the potential to affect the population density and community structure of prey mollusks in recipient coastal ecosystems through predation.     

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.     

Immunomodulatory effects of <Emphasis Type="Italic">Rhodomyrtus tomentosa</Emphasis> leaf extract and its derivative compound,rhodomyrtone, on head kidney macrophages of rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>)

Rhodomyrtus tomentosa is a medicinal plant that shows biological effects including immunomodulatory activity on human and other mammals but not in fish. In this study, we evaluated the in vitro immunomodulatory effects of R. tomentosa leaf extract and its active compound, rhodomyrtone, on the immune responses, using rainbow trout (Oncorhynchus mykiss) head kidney (HK) macrophages as a model. The tested immune functions included the expression of genes involved in innate immune and inflammatory responses and the production of reactive oxygen species (ROS). Gene expression was evaluated after exposure to 10 μg mL^?1 of R. tomentosa and 1 μg mL^?1 of rhodomyrtone for 4 and 24 h. R. tomentosa and rhodomyrtone induced changes in the expression of pro-inflammatory cytokines (il1β, il8, and tnfα), anti-inflammatory cytokines (il10 and tgfβ), inducible enzymes (inos, cox2, and arginase), and an antioxidant enzyme (gpx1). Co-exposure of R. tomentosa with LPS resulted in a prominent reduction in the expression of genes related to an inflammatory process (il1β, il8, tnfα, inos, saa, hepcidin, and gpx1), suggesting anti-inflammatory effects. Similarly, co-exposure of rhodomyrtone with LPS led to a downregulation of inflammation-related genes (il1β, inos, saa, and hepcidin). In addition, exposure to both natural plant products caused a reduction in cellular ROS levels by HK macrophages. The present results indicate that R. tomentosa and rhodomyrtone exerted immunostimulatory and anti-inflammatory effects on fish macrophages, thus opening up the possibility of using these natural products to further develop immunostimulants for health management in aquaculture.     

New insights into the evolution,hormonal regulation,and spatiotemporal expression profiles of genes involved in the Gfra1/Gdnf and Kit/Kitlg regulatory pathways in rainbow trout testis

The present study aimed to investigate whether the Gfra1/Gdnf and/or Kit/Kitlg regulatory pathways could be involved in the regulation of spermatogonial cell proliferation and/or differentiation in fish. Homologs of the mammalian gfra1, gdnf, kitr, and kitlg genes were identified in gnathostomes and reliable orthologous relationships were established using phylogenetic reconstructions and analyses of syntenic chromosomal fragments. Gene duplications and losses occurred specifically in teleost fish and members of the Salmoninae family including rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar). Some duplicated genes exhibited distinct spatiotemporal expression profiles and were differently regulated by hormones in rainbow trout. Undifferentiated type A spermatogonia expressed higher levels of kitrb and kitra2 making them possible target cells for the gonadal kitlgb and somatic kitlga before the onset of spermatogenesis. Interestingly, gdnfa and gdnfb ohnologous genes were poorly expressed before the onset of spermatogenesis. The expression level of gdnfb was correlated with that of the vasa gene suggesting that the late increased abundance of gdnfb during spermatogenesis onset was due to the increased number of spermatogonial cells. gfra1a2 was expressed in undifferentiated type A spermatogonia whereas gfra1a1 was mainly detected in somatic cells. These observations indicate that the germinal gdnfb ligand could exert autocrine and paracrine functions on spermatogonia and on testicular somatic cells, respectively. Fsh and androgens inhibited gfra1a2 and gdnfb whereas gfra1a1 was stimulated by Fsh, androgens, and 17α, 20β progesterone. Finally, our data provide evidences that the molecular identity of the male germ stem cells changes during ontogenesis prior to spermatogenesis onset.     

The changes in cortisol and expression of immune genes of GIFT tilapia <Emphasis Type="Italic">Oreochromis niloticus</Emphasis> (L.) at different rearing densities under <Emphasis Type="Italic">Streptococcus iniae</Emphasis> infection

The present study was conducted to investigate the effects of stocking density on serum cortisol (COR) levels and expression of immune genes in the head kidney of juvenile GIFT tilapia (Oreochromis niloticus) after Streptococcus iniae (S. iniae) infection. Juveniles (2700) were distributed into 30 tanks at five stocking densities (150, 300, 450, 600 and 750 g/m³), and each treatment had six replicates. After a 45-day feeding trial, a S. iniae challenge study was conducted for 96 h. Differences in mortality, serum COR and gene expression of C-type lysozyme, heat-shock protein 70 (HSP70), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) were analyzed. The levels of serum COR at all infected treatments were significantly higher compared with the levels of pre-infection (P < 0.05). At 96 h post-infection, serum COR levels of high stocking densities (600 and 750 g/m³) were significantly higher than those of low-density groups (P < 0.05); the mRNA levels of C-type lysozyme, HSP70, IL-1β, TNF-α and IFN-γ were significantly lower in tilapia reared at 600 g/m³ group than in those reared at 300 g/m³ group (P < 0.05). Fish reared in high-density treatments (600 and 750 g/m³) exhibited significantly higher post-challenge mortality (43.3 and 40.0 %) at 96 h following S. iniae infection, and it was significantly higher than other groups (P < 0.05). The data suggest that when living in high stocking density, GIFT tilapia showed decrease in immune capability, together with increased serum COR and susceptibility to S. iniae.     

Metagenomic analysis using 16S ribosomal RNA genes of a bacterial community in an urban stream,the Tama River,Tokyo

In an effort to determine genus- or species-level taxonomic profiles and diversity of bacterial consortia in the Tama River around urban Tokyo, next-generation sequencing technology targeting a 16S ribosomal RNA (rRNA) gene amplicon was employed. Metagenomic analysis performed by an Ion Personal Genome Machine after sequentially filtering samples through 5-, 0.8- and 0.2-μm filters yielded 1.48 Gb of 16S sequences (average 2.38 M reads/sample). The results indicated that half of the bacterial sequences belonged to Proteobacteria, followed by Bacteroidetes, Actinobacteria and Cyanobacteria. Flavobacterium (Bacteroidetes), possibly including a potential fish pathogen, was the most numerous genera in the Tama River metagenome, and accounted for?~?16% of assigned 16S reads, followed by Mycobacterium. Other dominant bacterial genera including Zoogloea, Sediminibacterium, Hyphomicrobium, Sphingopyxis, Thiothrix and Lysobacter, were thought to be associated with waste water and sludge. MiSeq metagenomic analysis revealed that environmental factors, particularly water temperature, influenced the bacterial composition throughout the year, with a strong negative correlation observed for Proteobacteria and a positive correlation for Bacteroidetes. In terms of bacterial genera, Flavobacterium was positively correlated with temperature, while Polaromonas, Pseudomonas and Bradyrhizobium were negatively correlated with this, suggesting dynamic change in the free-living bacterial population throughout the year and versatile adaptation strategies in relation to environmental factors.     

Characterization of a low-density lipoprotein receptor,Lrp13, in Chinese tongue sole (<Emphasis Type="Italic">Cynoglossus semilaevis</Emphasis>) and medaka (<Emphasis Type="Italic">Oryzias latipes</Emphasis>)

As an important economic marine species cultured in China, Chinese tongue sole (Cynoglossus semilaevis) has interested us due to its sexual dimorphism and ZW/ZZ sex determination system. In a previous study, dmrt1 was identified as a dosage-dependent male-determining gene. In the present study, a female-specific expressed gene, cse0440, initially annotated as lrp1b-like, was identified from chromosome W of C. semilaevis. In view of the differences between cse0440 and lrp1b in terms of expression pattern, a phylogenetic analysis containing 85 LRP proteins was constructed and provided an evidence to re-annotate cse0440 as cseLRP13. In addition, two orthologues of cseLRP13 were separately identified from W and Z chromosomes: cseLRP13-W and cseLRP13-Z. The subsequent multiple sequence alignment and syntenic arrangements of LRP13 in C. semilaevis, Japanese medaka (Oryzias latipes), large yellow croaker (Larimichthys crocea), striped bass (Morone saxatilis), white perch (Morone americana) and Fugu rubripes (Takifugu rubripes) further supported this re-annotation. RT-PCR and in situ hybridization revealed that cselrp13 was exclusively expressed in the oocytes and follicles of ovaries. These results suggested that lrp13 may play important roles in female reproduction. In future, with the advancement of micromanipulation in flatfish, the detailed function of two lrp13 orthologues in C. semilaevis will be elucidated.     

Growth performance and biochemical composition of juvenile pearl oyster <Emphasis Type="Italic">Pinctada martensii</Emphasis> fed on artificial diets

The effects of three artificial diets (S1, S2 and S3) on survival, growth and biochemical composition of one-year-old pearl oyster Pinctada martensii were investigated. Six experimental groups (EG1, EG2, EG3, EG4, EG5 and EG6) and one control group (CG) were set up. EG1, EG2 and EG3 were solely fed on S1, S2 and S3, respectively. EG4, EG5 and EG6 were fed on mixed diets, as follows: S1 and Platymonas subcordiformis; S2 and P. subcordiformis; and S3 and P. subcordiformis, respectively. CG was fed on only P. subcordiformis. All groups were continuously fed for 60 days. Survival, growth and biochemical composition of soft tissues were compared across the groups. Results showed that survival rate, the absolute growth rate (AGR) and relative growth rate (RGR) of shell length did not differ significantly across the groups (p > 0.05). The AGR and RGR of total weight differed significantly among the groups (p < 0.05). AGR and RGR of shell length and total weight were the highest in EG5 and the lowest in EG1. Gross fat content showed insignificant differences among the groups (p > 0.05). However, gross protein content and ash content showed significant differences across the groups (p < 0.05). The gross protein of the groups solely fed on artificial diets was lower than those of the groups fed on mixtures of artificial diets and microalgae or single microalgae. The contents of other amino acids, total amino acids (TAAs) and essential amino acids (EAAs) showed significant differences across the groups (p < 0.05). TAA, EAA and delicious amino acids of the groups fed solely on artificial diets were lower than those of the groups fed on mixtures of artificial diets and microalgae or single microalgae. Results indicated that the artificial diet (S2) can serve as substitutes of microalgal diets for P. martensii.     

Effect of the early temperature on the growth of larvae and postlarvae turbot, <Emphasis Type="Italic">Scophthalmus maximus</Emphasis> L.: muscle structural and ultrastructural study

Turbot specimens were kept at three temperatures (T _s ): warm (W) (21–22 °C), ambient (A) (17–18 °C) and cold (C) (13–14 °C) during the larval and early postlarval stages. At 90 days posthatching (dph), all of them were transferred to ambient T until 190 dph. At 2–3 dph, the specimens showed a monolayer of red muscle and immature white fibres; external or dermomyotome cells (presumptive myogenic cells) were observed on the surface of the red muscle. In the following stages, many myogenic cells and presumptive myogenic precursors were observed within the myotome, presumably derived of the dermomyotome. When comparing the growth at the same age (2, 10, 25, 37 dph), the body length and the muscle growth were positively influenced by the warm T, being the hyperplasia the muscle parameter more significantly influenced. The development rate was also positively correlated with the high T: the beginning of the metamorphosis took place at 15, 23 and 25 dph at W, A and C temperatures, respectively, with the highest body length values at ambient temperature. The metamorphosis finished at 25, 30 and 37 dph at W, A and C temperatures, respectively, with the highest body length values at warm temperature. However, the muscle cellularity was similar in all the groups at the end of the metamorphosis. At 90 and 190 dph, the largest body length was observed at W temperature. However, the muscle cellularity was similar between A and W; the number of fibres was similar in all the groups at 190 dph, which shows the beginning of a compensatory muscle growth in A and C, mainly in A.