Lagged recovery of fish spatial distributions following a cold-water perturbation.

Anomalous local temperature and extreme events (e.g. heat-waves) can cause rapid change and gradual recovery of local environmental conditions. However, few studies have tested whether species distribution can recover following returning environmental conditions. Here, we tested for change and recovery of the spatial distributions of two flatfish populations, American plaice (Hippoglossoides platessoides) and yellowtail flounder (Limanda ferruginea), in response to consecutive decreasing and increasing water temperature on the Grand Bank off Newfoundland, Canada from 1985 to 2018. Using a Vector Autoregressive Spatiotemporal model, we found the distributions of both species shifted southwards following a period when anomalous cold water covered the northern sections of the Grand Bank. After accounting for density-dependent effects, we observed that yellowtail flounder re-distributed northwards when water temperature returned and exceeded levels recorded before the cold period, while the spatial distribution of American plaice has not recovered. Our study demonstrates nonlinear effects of an environmental factor on species distribution, implying the possibility of irreversible (or hard-to-reverse) changes of species distribution following a rapid change and gradual recovery of environmental conditions.

Importance of experienced thermal history: Effect of acclimation temperatures on the high-temperature tolerance and growth performance of juvenile marbled flounder.

Experienced thermal history often affects the temperature tolerance of fish; however, the effect of thermal history on growth performance is unclear. To contribute to effective stocking (release of hatchery-reared juveniles in the field), we conducted four laboratory experiments using juvenile marbled flounder (Pseudopleuronectes yokohamae, around 30 mm standard length and 0.3 g body wet weight) acclimated at 12 °C and 24 °C for approximately 2 weeks to investigate the effects of acclimation temperature on high-temperature tolerance, food consumption, and growth performance. The acclimation to 24 °C increased tolerance to high temperatures, as shown in a 24-h exposure experiment and in a temperature elevation experiment. The 50% lethal temperature (upper incipient lethal temperature) was estimated to be 25.9 °C and 29.0 °C for the 12 °C and 24 °C acclimation groups, respectively. In subsequent experiments, we tested the effects of high and low temperature acclimation on the food consumption and growth performance of two size groups of juveniles (28.7 ± 2.0 and 34.5 ± 2.9 mm, mean ± SD), that were reared at temperatures ranging from 14 °C to 23 °C. The optimal temperature for growth was 20 °C and did not differ between the acclimation temperatures or between the size groups. However, food consumption and growth performance were suppressed by acute temperature changes. Specifically, feeding and growth were lower in the 24 °C-acclimated group than in the 12 °C-acclimated group when exposed to 14 °C, which is close to the natural water temperature at release in the field. These results suggest that experienced thermal history does not affect the optimal temperature but can affect the growth performance of juveniles. To maximize the post-release growth of hatchery-reared juveniles, the influence of thermal history should be taken into consideration and acute thermal changes before release should be avoided.

Dietary Supplementation of Bacillus sp. SJ-10 and Lactobacillus plantarum KCCM 11322 Combinations Enhance Growth and Cellular and Humoral Immunity in Olive Flounder (Paralichthys olivaceus).

Experiments were conducted to identify different ratios of Bacillus sp. SJ-10 and Lactobacillus plantarum KCCM 11322 mixtures at a concentration of 1 × 108 CFU/g diet; the effects on growth and cellular and humoral immune responses and the characteristics of disease protection in olive flounder (Paralichthys olivaceus). Flounder were divided into six groups and fed control diet D-1 (without Bacillus sp. SJ-10 and L. plantarum KCCM 11322), positive control diets D-2 (Bacillus sp. SJ-10 at 1 × 108 CFU/g feed) and D-3 (L. plantarum KCCM 11322 at 1 × 108 CFU/g feed); or treatment diets D-4 (3:1 Bacillus sp. SJ-10 and L. plantarum KCCM 11322 at 0.75 + 0.25 × 108 CFU/g feed), D-5 (1:1 Bacillus sp. SJ-10 and L. plantarum KCCM 11322 at 0.50 + 0.50 × 108 CFU/g feed), or D-6 (1:3 Bacillus sp. SJ-10 and L. plantarum KCCM 11322 at 0.25 + 0.75 × 108 CFU/g feed) for 8 weeks. Group D-4 demonstrated better growth and feed utilization (P < 0.05) compared with the controls and positive controls. Similar modulation was also observed in respiratory burst for all treatments and in the expression levels of TNF-α, IL-1ß, IL-6, and IL-10 in different organs in D-4. D-4 and D-5 increased respiratory burst, superoxide dismutase, lysozyme, and myeloperoxidase activities compared with the controls, and only D-4 increased microvilli length. When challenged with 1 × 108 CFU/mL Streptococcus iniae, the fish in the D-4 and D-5 groups survived up to 14 days, whereas the fish in the other groups reached 100% mortality at 11.50 days. Collectively, a ratio-specific Bacillus sp. SJ-10 and L. plantarum KCCM 11322 mixture (3:1) was associated with elevated growth, innate immunity, and streptococcosis resistance (3:1 and 1:1) compared with the control and single probiotic diets.

Transcriptome Profiling Reveals the Sexual Dimorphism of Gene Expression Patterns during Gonad Differentiation in the Half-Smooth Tongue Sole (Cynoglossus semilaevis).

The half-smooth tongue sole (Cynoglossus semilaevis), one of the most economically-important fish species in China, exhibits sexually dimorphic growth. An understanding of sex-related gene expression patterns in the tongue sole may inform sex regulation and breeding processes that increase fish production. However, the gene expression patterns during gonad development in the tongue sole remain unknown. In this study, transcriptome sequencing analyses were performed during gonad differentiation in the tongue sole, namely, at 62 days post-hatching (dph), 100 dph, 120 dph, and 150 dph. Differentially expressed genes associated with sex differentiation and gonad development were identified at each time point. Trend analysis showed that gene expression patterns varied over time. These expression patterns either explained common, non-sexually-dimorphic features or indicated significant sexual dimorphism. Transcript structure analyses identified both sex and time differences among samples. This study investigated the time-dependent expression patterns of several sex-related genes, including Dmrt1, Amh, Foxl2, aromatase encoding gene, Esr, and the Sox gene family, during gonad differentiation in the tongue sole. These results might clarify the significant sexual differences during early development in the tongue sole and might provide insight into the mechanisms controlling sex differentiation and development.

Evaluating socio-economic and conservation impacts of management: A case study of time-area closures on Georges Bank.

Globally, economies and marine ecosystems are increasingly dependent on sustainable fisheries management (SFM) to balance social, economic, and conservation needs. The overarching objectives of SFM are to maximize both conservation and socio-economic benefits, while minimizing short-term socio-economic costs. A number of tools have been developed to achieve SFM objectives, ranging from fishery specific to ecosystem-based strategies. Closures are a common SFM tool used to balance the trade-off between socio-economic and conservation considerations; they vary in scope from small-scale temporary closures to large-scale permanent networks. Unfortunately, closures are frequently implemented without a plan for monitoring or assessing whether SFM objectives are met. In situations in which a monitoring plan is not in place we propose that commonly available fishery data can often be used to evaluate whether management tools are effective in meeting SFM objectives. Here, we present a case study of closures on Georges Bank that shows how fishery data can be analyzed to perform such an assessment. Since 2006, on the Canadian side of Georges Bank, seasonal scallop fishery closures have been implemented with the aim of reducing by-catch of Atlantic cod (Gadus morhua) and yellowtail flounder (Pleuronectes ferruginea) during spawning. In lieu of data from a dedicated monitoring program, we analyzed data from Vessel Monitoring Systems (VMS), fishery logbooks, and a scallop survey to assess the impact of these closures on the scallop fishery, and use observer data (i.e. by-catch) to assess the effectiveness of these closures in meeting their conservation objective. While compliance for these time-area closures was high, the closures did not significantly displace fishing activity and overall there was limited evidence of an impact on the scallop fishery. Further, the discard rates for both cod and yellowtail were above average when their respective closures were active. These results suggest that improvements to the closures design and/or other measures may be required to achieve the desired SFM objectives.

A comprehensive description of oocyte developmental stages in Pacific halibut, Hippoglossus stenolepis.

Accurate characterization of oocyte development is essential to understanding foundational aspects of reproductive biology and successful management of Pacific halibut (Hippoglossus stenolepis). Here this study provides complete histological descriptions for eight oocyte developmental stages in addition to postovulatory follicles and demonstrates the potential for oocyte size frequency distribution to act as a proxy for ovarian developmental stage and future maturity assessments. Importantly, it provides the first histological evidence that Pacific halibut have a group-synchronous ovarian developmental pattern with determinate fecundity and support for their batch-spawning strategy.

miR-430a regulates the development of left-right asymmetry by targeting sqt in the teleost.

microRNAs (miRNAs) are short, endogenous non-coding RNAs that contain approximately 18-22 nucleotides. miRNAs are involved in gene regulation by recognizing and binding the 3'UTR of target gene. In our previous data, miR-430 family showed significant differential expression modes through metamorphosis in Japanese flounder. It was speculated that miR-430a plays a key role in left-right patterning. We predicted the targets of miR-430a and gene ontology (GO) was performed. We speculated miR-430a is involved in the basal molecular function and organ development. In Japanese flounder, sqt as a target of miR-430a was enriched into heart development term. Sqt has been reported to participate in mesendoderm formation and organ development. Cardiac morphogenesis is the first asymmetric development process, which breaks left-right symmetry in bilateria. It was used as a marker to detect L-R asymmetric effects of miR-430a. Overexpression and suppression of miR-430a resulted in abnormal KV (Kupffer's vesicles) development and disordered in nodal-related expression with consequent cardiac laterality. Squint mRNA of Japanese flounder (Posqt) as a target of miR-430a was overexpressed and caused similar phenotype with miR-430a suppression group, such as longer cilia in KV and high range of clmc2 and spaw ectopic expression. Moreover, rescue experiments were performed and suggested that cardiac and KV defections, induced by overexpressing miR-430a, could be rescued by injecting Posqt mRNA. These results suggested that miR-430a regulates the development of left-right asymmetry by targeting sqt in the teleost.

Probiotic effects of mixture of Groenewaldozyma salmanticensis and Gluconacetobacter liquefaciens on growth and immune responses in Paralichthys olivaceus.

This study was performed to evaluate the effects of dietary probiotics on growth, non-specific immune responses and disease resistance in olive flounder, Paralichthys olivaceus. During 8 weeks, the fish were fed the five experimental diets such as a basal commercial diet (CON), oxytetracycline (OTC) and three basal diets containing Bacillus subtilis (BS), a commercial microbial product (CES) and a mixture of yeast and bacterium (PI), respectively. Fish fed all the probiotics diets and OTC showed a significantly higher growth than fish-fed CON (P < 0·05). Fish-fed PI had a significantly higher nitroblue tetrazolium activity, whereas fish-fed CES showed a higher lysozyme level (P < 0·05). A 7-day challenge test also showed that fish-fed PI had a cumulative survival rate equivalent to that of fish-fed OTC (P < 0·05). Moreover, the diet (PI) appeared to increase the diversity of microbial community in the fish. All these results suggest that the probiotics diet could function as a potential antibiotic replacer in the olive flounder. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is unique in revealing that a diet mixture of yeast, Groenewaldozyma salmanticensis and bacterium Gluconacetobacter liquefaciens can enhance growth, innate immunity and diversity of microbial community including dominant species in the olive flounder. All these indicate that the diet mixture could function as a potential antibiotic replacer in one of the most commercially important fisheries in South Korea.

Dietary effect of low fish meal aquafeed on gut microbiota in olive flounder (Paralichthys olivaceus) at different growth stages.

This study was conducted to investigate the long-term effect of a low fish meal (FM) diet comprising plant-based protein sources (PPS) on changes of gut microbial diversity in olive flounder (Paralichthys olivaceus) over the course of life. Two experimental diets were prepared to contain 74% FM (control) or 52% FM with 22% PPS (30% FM replacement, FM30). Fish were fed one of the two experimental diets for 8 months, and we collected the midgut contents to analyze the gut bacterial community by Illumina MiSeq based on the metagenomic sequences in the V3-V4 regions of 16S rRNA. We found that there were nine dominant phyla, which in turn presented Proteobacteria, Firmicutes, and Actinobacteria as the three major phyla in the gut microbiota of the flounder. At genus level, the dominant genera were Delftia, Prevotella, and Chthoniobacter at the juvenile stage (below 100 g/fish); Chthoniobacter, Bacillus, and Bradyrhizobium at the grower stage (400 g/fish); Chthoniobacter, Bacillus, and Delftia at the subadult stage (800 g/fish); and Lactobacillus and Prevotella at the adult stage (over 1,000 g/fish). The microbial diversity in olive flounders arched from the juvenile and subadult stage and reached a plateau thereafter. The fish fed the FM30 diet significantly had an increased abundance of Lactobacillus and Photobacterium and had less abundance of Prevotella and Paraprevotella than the control. However, the effect of dietary PPS was not significant on total microbial richness, indicating no negative effect as feed sources on the intestinal microbiota in olive flounder. These results indicate that the life stage of olive flounder is more important in modulating intestinal microbiota than is the diet. It could also be concluded that dietary PPS might be used as a potential fish meal alternative without any compromising effects on microbial diversity of olive flounder for long-term feeding.

An hypoxia-tolerant flatfish: consequences of sustained stress on the slender sole Lyopsetta exilis (Pleuronectidae) in the context of a changing ocean.

Slender sole Lyopsetta exilis is an abundant groundfish on the continental shelf and inner waters of British Columbia, Canada, where it reaches a maximum standard length of 44 cm. Benthic image surveys coupled with oxygen measurements in Saanich Inlet document a dense population in bottom conditions near anoxia (0.03 ml l-1 oxygen) where diel migrating zooplankton intersect the bottom; we confirm this species is a planktivore, which limits its depth range to the base of the migration layer. In a comparison with slender sole from a nearby well-oxygenated habitat, several probable effects of living in severe hypoxia emerge: both sexes are significantly smaller in Saanich and the sex ratio is male-skewed. Otoliths from the Saanich fish were difficult to read due to many checks, but both sexes were smaller at age with the largest female (20 cm) from the hypoxia zone registering 17 years. Hypoxia appears to have a direct consequence on growth despite good food supply in this productive basin. Hyperventilation, a low metabolic rate and a very low critical oxygen tension help this fish regulate oxygen uptake in severely hypoxic conditions; it will be particularly resilient as the incidence of hypoxia increases on the continental shelf. Data from small-mesh bottom-trawl surveys over four decades reveal an increase in mean annual catch per unit effort in southern regions of the province, including the outer shelf and the Strait of Georgia. The California Cooperative Oceanic Fisheries Investigations (CalCOFI) ichthyoplankton database records a general decline in fish larvae on the Oregon-California shelf since 1990, but slender sole larvae are increasing there, as they are in the Strait of Georgia. We project that the slender sole will gain relative benefits in the future warming, deoxygenated northeast Pacific Ocean.

Analyses of mRNA-seq and miRNA-seq of the brain reveal the sex differences of gene expression and regulation before and during gonadal differentiation in 17ß-estradiol or 17α-methyltestosterone-induced olive flounder (Paralichthys olivaceus).

Olive flounder (Paralichthys olivaceus) is a commercially important flatfish species cultured in East Asia. Female flounders generally grow more rapidly than males, therefore control of the sex ratio seems to be a proposed way to increase production. However, the sex determination gene and sex determination mechanism have yet been elucidated. The brain is an important organ that is involved in gonadal development. To explore the sex differences of gene expression in the brain before and during the flounder gonadal differentiation, we used messenger RNA (mRNA)-seq technology to investigate transcriptomes of male and female brains. Between female and male brains, 103 genes were differentially expressed before ovarian differentiation, 16 genes were differentially expressed before testicular differentiation, and 64 genes were differentially expressed during gonadal differentiation. According to annotation and Kyoto Encyclopedia of Genes and Genomes information, the differentially expressed genes (DEGs) were involved in circadian rhythm, circadian rhythm-fly, circadian entrainment, dopaminergic synapse, calcium signaling, glutamatergic synapse, taste transduction, herpes simplex infection, long-term depression, retrograde endocannabinoid signaling, and the synaptic vesicle cycle pathways. MicroRNA (miRNA)-seq was performed during the gonadal differentiation and the target genes of miRNAs were predicted. Integrated analysis of mRNA-seq and miRNA-seq showed that 29 of the 64 DEGs were regulated by the differentially expressed miRNAs during the gonadal differentiation. Our study provides a basis for further studies of brain sex differentiation and the molecular mechanism of sex determination in olive flounder.

The role of miR-92 in regulating early development and metamorphosis of Japanese flounder Paralichthys olivaceus.

MicroRNAs are a class of short non-coding RNAs that contain approximately 22 nucleotides and play a regulatory role in RNA silencing and translational repression. miR-92 belongs to the miR-17-92 family and has a regulatory effect on cell proliferation, apoptosis, and expression of proto-oncogenes and tumor suppressor genes. However, its function in flatfish is unclear. In this study, we used farmed Japanese flounder, Paralichthys olivaceus, and showed that gata5 is a target gene of miR-92. Experiments on miR-92 overexpression indicated that gata5 and sox17 were downregulated, while the transcription level of ntl increased. By contrast, depletion of miR-92 resulted in increased gata5 and sox17 levels and reduced ntl level. Moreover, thiourea treatment indicated that miR-92 may inhibit the metamorphic development of Japanese flounder. Our study suggests that miR-92 regulates the fate of endoderm and mesoderm by controlling gata5.

Evolutionary significance and regulated expression of Tdrd family genes in gynogenetic Japanese flounder (Paralichthys olivaceus).

Tudor domain-containing proteins (TDRDs) are highly conserved among organisms and have a function in gonads to regulate gametogenesis and genome stability through the piwi-interacting RNA (piRNA) pathway. With diverse sexual development patterns in teleost species, the evolution and function of Tdrd genes among teleosts remain unclear. Here, we identified and characterized 12 Tdrd genes (PoTdrds) in Japanese flounder (Paralichthys olivaceus) which represents dramatic sexual dimorphic metrics and sex reversal during sex differentiation. Phylogenetic and comparative synteny indicated the gain and loss of Tdrd genes after teleost-specific whole-genome duplication (3R-WGD). Tdrd1, Tdrd5, Tdrd6 and Ecat8 were abundantly expressed in their gonads. Four PoTdrds (Tdrd6, Tdrd7b, Tdrd9 and Ecat8) represented significant male-biased expression in gynogenetic and wild-type Japanese flounder gonads (p < .01). This finding indicated their important roles in spermatogenesis of P. olivaceus. Some PoTdrds were either highly up-regulated in gynogenetic testis (Tdrd3, Tdrd5, Tdrd7b and Ecat8) or down-regulated in gynogenetic ovary (Tdrkh, Tdrd3, Tdrd6l) compared with wild-type gonads (p < .05). Molecular evolution tests revealed that the selective pressure of Tdrd6/6l differed between ancestral aquatic and terrestrial organisms with 13 positively selected sites found in the ancestral lineages of teleost Tdrd6. Expression profile analysis suggested that PoTdrd6 differed significantly from PoTdrd6l, indicating sub-functionalization after 3R-WGD. All these results are important for the functional annotation of Tdrd genes and can benefit the further deciphering of Tdrd functions during gonadal development and gametogenesis of teleost fish.

Novel breeding approach for Japanese flounder using atmosphere and room temperature plasma mutagenesis tool.

BACKGROUND: Artificial induction of mutagenesis is effective for genetic resource innovation and breeding. However, the traditional mutation methods for fish breeding are not convenient or safe for daily use. Hence, development of a simple, safe and effective mutagenesis method with a high mutation rate and applicability to multiple fish species, is needed. RESULTS: We reported the first successful mutagenesis in a marine aquaculture fish species, Japanese flounder, Paralichthys olivaceus, using a novel atmosphere and room temperature plasma (ARTP) mutagenesis tool. ARTP treatment time was optimized for the fertilized eggs and sperm, respectively. Eggs fertilized for 60 min were treated by ARTP with a radio-frequency power input of 120 W, and the ARTP treatment time was 25 min. Under an ARTP radio-frequency power input of 200 W, the optimal treatment time for sperm diluted with Ringer's solution by 1:40 v/v was 10 min. The ARTP-treated group presented differences in morphological traits such as body height, total length among individuals at day 90 after hatching. Whole-genome sequencing was used to reveal the mutation features of ARTP-treated individuals collected at day 120 after hatching. In total, 69.25Gb clean data were obtained from three controls and eight randomly selected ARTP-treated individuals, revealing 240,722 to 322,978 SNPs and 82,149 to 86,798 InDels located in 17,394~18,457 and 12,907~13,333 genes, respectively. The average mutation rate reached 0.064% at the genome level. Gene ontology clustering indicated that genes associated with cell components, binding function, catalytic activity, cellular process, metabolic process and biological regulation processes had higher mutation rates. CONCLUSIONS: ARTP mutagenesis is a useful method for breeding of fish species to accelerate the selection of economically important traits that would benefit the aquaculture industry, given the variety of mutations detected.

Methylation status and expression patterns of myomaker gene play important roles in postnatal development in the Japanese flounder (Paralichthys olivaceus).

Myomaker is a membrane protein that plays a crucial role in the fusion of myoblasts during muscle growth. DNA methylation, a significant factor, regulates gene expression. The aim of this study was to examine the methylation and mRNA expression patterns of the myomaker gene during 8 different postnatal developmental stages in the Japanese flounder (L: 7 days post hatch (dph); M1: 21 dph; M2: 28 dph; M3: 35 dph; J1: 90 dph; J2: 180 dph; A1: 24 months; A2: 36 months). Muscle tissue samples were taken from Japanese flounder at different postnatal development stages to measure the extent of DNA methylation and gene expression. Methylation level in the promoter and exon 1 of myomaker was measured using bisulfite sequencing, and the relative expression of myomaker during each developmental stage was measured by quantitative PCR. The relative expression levels of myomaker were up-regulated from stages L to M2, M3 to J2, and methylation of myomaker was negatively correlated with mRNA expression. Furthermore, the CpG site located at -26 bp in the promoter was the lowest methylated region in all developmental stages. These results offer a basis for understanding the mechanism by which myomaker regulates muscle formation during postnatal development.

Heat-killed Bacillus sp. SJ-10 probiotic acts as a growth and humoral innate immunity response enhancer in olive flounder (Paralichthys olivaceus).

Investigations were carried out to evaluate and quantify the effects of dietary supplementation with heat-killed (HK) Bacillus sp. SJ-10 (BSJ-10) probiotic (1 × 108 CFU g-1) on the growth and immunity of olive flounder (Paralichthys olivaceus). Flounder (averagely 9.64 g) were divided into two groups, and fed control and HK BSJ-10 (HKBSJ-10)-inoculated diets for 8 weeks. Investigations were carried out on growth and feed utilizations, innate immunity, serum biochemical parameters, microvilli length, and pro- and anti-inflammatory cytokine gene (tumor necrosis factor [TNF]-α, interleukin [IL]-1ß, IL-6, and IL-10) transcriptions. Compared to control, HKBSJ-10 diet significantly (P < 0.05) enhanced weight gain and protein efficiency ratio, 1.17 and 1.11 folds respectively. Humoral innate immune parameters, lysozyme and superoxide dismutase in treatment group were also elevated by 1.34 and 1.16 folds. Similarly, an increased (P < 0.05) relative expressions of TNF-α, IL-1ß, IL-6 were recorded in liver (2.71, 3.38, and 4.12 folds respectively), and gill (2.08, 1.98, and 1.81 folds respectively) than that of controls. Moreover, after challenge with Streptococcus iniae (1 × 108 CFU mL-1), the HKBSJ-10-fed group exhibited significantly higher protection (P < 0.05) against streptococcosis compared to controls, validating the observed changes in immune parameters and induction on the cytokine-encoding genes. Therefore, HKBSJ-10 increases growth, modulates innate immune parameters, and protects olive flounders against streptococcosis.

Roles of piwil1 gene in gonad development and gametogenesis in Japanese flounder, Paralichthys olivaceus.

PIWI family member piwil1, which associates with Piwi-interacting RNA (piRNA), is responsible in regulation of germ cell differentiation and maintenance of reproductive stem cells. In this study, we analyzed the piwil1 gene in Paralichthys olivaceus. Bioinformatics analysis and structure prediction showed that piwil1 had the conserved domains: PAZ domain and PIWI domain. Expression analysis during embryonic development implied that piwil1 gene was maternally inherited. The tissue distribution showed a sexually dimorphic gene expression pattern, with higher expression level in testis than ovary. In situ hybridization results demonstrated that piwil1 was predominantly distributed in oogonia, oocytes, sertoli cells and spermatocytes. A CpG island was predicted in the 5'-flanking region of piwil1 gene, and its methylation levels showed significant disparity between males and females, indicating that the sexually dimorphic expression of piwil1 gene might be regulated by methylation. Furthermore, we explored the distinct roles of human chorionic gonadotropin and 17α-methyltestosterone in regulating the expression of piwil1, and found that piwil1 was interacting with the HPG axis hormones. These results indicated that piwil1 might play a crucial role in gonadal development and gametogenesis in Paralichthys olivaceus.

The evolution and possible role of two Sox8 genes during sex differentiation in Japanese flounder (Paralichthys olivaceus).

Sox8 genes, as members of the Sox family, have been studied widely in mammals. However, regulation of sox8 genes in teleosts has rarely been studied, and functional analysis of these genes in teleosts has rarely been performed. Here, two duplicates of sox8 genes were identified in Japanese flounder, Posox8a and Posox8b. The analysis of expression showed that Posox8a and Posox8b were expressed in Sertoli cells of the testis, indicating that they play important roles in development and functional maintenance of the testis. Positive selection and phylogenetic analysis found that both Posox8a and Posox8b underwent the purification selection during evolutionary and that sox8 was most likely to be the ancestor sox8a. These results suggested that both Posox8a and Posox8b had important biological functions after generation from three rounds of whole-genome duplication in Japanese flounder. The functional differentiation of Posox8a and Posox8b was verified using cell transfection and dual-luciferase reporter assays; Posox8a overexpression-promoted 3ß-hydroxysteroid dehydrogenase expression and Posox8b overexpression-promoted cytochrome P450 aromatase (cyp19a1; P450arom) expression. Finally, combined with Posox8a and Posox8b expression analysis from 30 to 100 days after hatch, we speculated that Posox8a and Posox8b might participate in the process of sex differentiation and gonadogenesis by regulating sex hormone biosynthesis in the Japanese flounder. Our study is the first to demonstrate the possible mechanism of Posox8a and Posox8b in Japanese flounder sex differentiation and gonadogenesis, laying a solid foundation for functional studies of sox8 genes in teleosts.

Effects of Winter Flounder Antifreeze Protein on the Growth of Ice Particles in an Ice Slurry Flow in Mini-Channels.

The control of ice growth in ice slurry is important for many fields, including (a) the cooling of the brain during cardiac arrest, (b) the storage and transportation of fresh fish and fruits, and (c) the development of distributed air-conditioning systems. One of the promising methods for the control is to use a substance such as antifreeze protein. We have observed and report here growth states of ice particles in both quiescent and flowing aqueous solutions of winter flounder antifreeze proteins in mini-channels with a microscope. We also measured ice growth rates. Our aim was to improve the levels of ice growth inhibition by subjecting the antifreeze protein solution both to preheating and to concentrating by ultrafiltration. We have found that the ice growth inhibition by the antifreeze protein decreased in flowing solutions compared with that in quiescent solutions. In addition, unlike unidirectional freezing experiments, the preheating of the antifreeze protein solution reduced the ice growth inhibition properties. This is because the direction of flow, containing HPLC6 and its aggregates, to the ice particle surfaces can change as the ice particle grows, and thus the probability of interaction between HPLC6 and ice surfaces does not increase. In contrast to this, ultrafiltration after preheating the solution improved the ice growth inhibition. This may be due to the interaction between ice surfaces and many aggregates in the concentrates.

Effects of green light on the growth of spotted halibut, Verasper variegatus, and Japanese flounder, Paralichthys olivaceus, and on the endocrine system of spotted halibut at different water temperatures.

We have previously shown that the somatic growth of barfin flounder, Verasper moseri, was promoted by green light. The present study was undertaken to elucidate whether growth-promoting effect of green light can be observed in other flatfishes and to understand the roles of endocrine systems in green light-induced growth. Herein, we demonstrated facilitation of growth by green light in the spotted halibut, Verasper variegatus, and Japanese flounder, Paralichthys olivaceus. Blue and blue-green light showed potencies that were similar to that of green light, while the potencies of red and white light were equivalent to that of ambient light (control). We also examined the effects of green light on growth and endocrine systems of V. variegatus at various water temperatures. Growth of the fish was facilitated by green light at four different water temperatures examined; the fish were reared for 31 days at 12 and 21 °C, and 30 days at 15 and 18 °C. Increase in condition factor was observed at 15 and 18 °C. Among the genes encoding hypothalamic hormones, expression levels of melanin-concentrating hormone 1 (mch1) were enhanced by green light at the four water temperatures. Expression levels of other genes including mch2 increased at certain water temperatures. No difference was observed in the expression levels of pituitary hormone genes, including those of growth hormone and members of proopiomelanocortin family, and in plasma levels of members of the insulin family. The results suggest that green light may generally stimulate growth of flatfishes. Moreover, it is conceivable that MCH, production of which is stimulated by green light, is a key hormone; it augments food intake, which is intimately coupled with somatic growth.