Effects of Ca and Mg concentrations in seawater on the growth and energy budget of juvenile Paralichthys olivaceus (Temminck & Schlegel, 1846)

Effects of seawater calcium (Ca) and magnesium (Mg) concentrations on the growth and energy budget of juvenile Paralichthys olivaceus (Temminck & Schlegel, 1846) were investigated at 20°C and a salinity of 30 psu. The Ca/Mg ratio (Ca : Mg = 1 : 3) in the experimental water was kept constant. Five treatments were set: C1, C2, C3, C4, and C5, and the Ca and Mg concentrations were 700, 1100, 1600, 2200, and 2800 (mg L^?1), respectively. After a 60‐day feeding trial the P. olivaceus survival rates were not affected significantly by Ca or Mg concentrations in the range of 700–2800 mg L^?1. However, the specific growth rates (SGR) and weight gain (WG) of all treatments decreased with ascending Ca and Mg concentrations. The Ca and Mg concentrations had a strong influence on various energy parameters, namely, energy deposited for growth (G), energy lost for respiration (R), and energy lost in excretion (U), but had little influence on energy lost in feces (F). This indicated that the growth rate differences under different concentrations of Ca and Mg mainly resulted from differences of assimilation efficiency and energy channeled into respiration. The highest net growth efficiency was attained at the lowest concentration of Ca and Mg in seawater, and the higher concentrations of Ca and Mg increased the energy channeled to respiration and excretion, which led to significant reduction in growth. Therefore, the optimal growth of P. olivaceus could be obtained by regulating the concentration of Ca and Mg at 700 mg L^?1, where the average energy budget was: 100C (100% energy consumed in food) = 47.21 (±0.63) G + 32.89 (±0.42) R + 16.71 (±0.56) F + 3.19 (±0.05) U.     

Effect of dietary fish oil replacement with palm oil on growth performance,hematology and liver anti‐oxidative enzymes of juvenile Japanese flounder Paralichthys olivaceus (Temminck & Schlegel, 1846)

A 60‐day feeding trial was conducted to evaluate the effects of dietary palm oil supplements on growth performances, hematology, liver anti‐oxidative enzymes and air exposure resistance of Japanese flounder, Paralichthys olivaceus (initial weights 2.56 ± 0.01 g). Five diets were tested wherein the dietary fish oil was replaced by palm oil at: 0% (Control), 20% (20%), 40% (40%), 50% (50%) and 60% (60%). After the feeding trial, the 20% dietary palm oil was shown to provide similar growth rates and feed efficiency with no negative effects compared to the control group (P > 0.05). Significantly lower growth rates and feed utilization were found in fish fed higher than 40% palm oil in the diet (P < 0.05). Except for total serum protein, the blood parameters, liver anti‐oxidative enzymes, stress resistance and proximate compositions of Japanese flounder were not altered, even with dietary palm oil up to 60% of the lipid source (P > 0.05). According to the present results, palm oil is a valuable lipid source substitute in Japanese flounder diets; around 20–40% fish oil can be replaced with palm oil with no negative effects.     

Survival,growth and physiological responses of juvenile Japanese flounder (Paralichthys olivaceus,Temminck & Schlegel, 1846) exposed to different dissolved oxygen concentrations and stocking densities

The aim of this study was to determine to what extent juvenile Japanese flounder can adapt to different stocking densities in captivity and to examine whether growth and some physiological parameters critical for welfare might be affected by different dissolved oxygen levels. Japanese flounder (Paralichthys olivaceus) juveniles (initial weight 1.27 ± 0.04 g/fish) were reared at five stocking densities (500, 1,000, 1,500, 2,000, 2,500 ind/m³) and two levels of dissolved oxygen (DO) concentration (5.5 ± 0.5 mg/L or 14 ± 2 mg/L) with duplicate tanks for each treatment in water‐recirculating systems for 40 days. Survival and activity of superoxide dismutase (SOD) were not affected either by stocking density or dissolved oxygen, whereas final body weight, specific growth rate (SGR) and feed conversion efficiency (FCE) of fish under low DO concentration decreased significantly with increasing stocking density. In contrast, growth of fish reared in high DO levels were unaffected by the stocking density. Furthermore, fish in this group had a higher feed intake and, consequently, grew faster (SGR) and achieved a higher final weight than fish reared at the low DO level. A significant reduction in hemoglobin (Hb) concentration and red blood cell (RBC) count of fish were recorded as DO concentration increased. Furthermore, the activity of protease decreased significantly with increasing stocking density and increased significantly with increasing DO concentration. The ventilation frequency results indicate that gill ventilation decreased significantly as DO levels increased. This study demonstrates that stocking density can affect the growth performance and physiological parameters critical for welfare of juvenile Japanese flounder. Also shown is that pure oxygen supplementation is an effective way to improve the growth of juvenile Japanese flounder when reared at a high stocking density.     

Compensatory growth of juvenile brown flounder Paralichthys olivaceus (Temminck & Schlegel) following thermal manipulation

The compensatory growth of juvenile brown flounder Paralichthys olivaceus (body mass c. 12 g) following different thermal exposure was investigated. Fish were exposed to one of the five temperatures: 8·5 ( T _8·5), 13·0 ( T _13·0), 17·5 ( T _17·5), 22·0 ( T _22·0) and 26·5° C ( T _26·5) for 10 days and fish grew best at 22·0° C. Then the water temperature in all treatments was equably adjusted to 22·0° C over 3 days. At the end of the following 30 days after temperature adjustment, there were no significant differences between body masses of fish in the different treatments (wet body mass at the end of the experiment ranged from 22·13 to 24·56 g). Results indicated that the juvenile P. olivaceus achieved complete compensatory growth. Analysis of the dynamics of the feeding rates and feed conversion efficiencies indicated that compensatory growth of the fish experienced low temperature ( T _8·5, T _13·5 and T _17·5) or high temperature ( T _26·5) exposure was mainly dependent on increasing feed intake (hyperphagia) and possibly by improvement in feed conversion efficiency. The moisture content was not affected by different temperature exposure significantly. The lipid and energy content of juvenile P. olivaceus in T _8·5, however, were significantly lower than other treatment. Results of the current study indicate that a short period of low or high temperature exposure may not affect annual growth, but may affect lipid and energy deposition.     

Japanese eel (Anguilla japonica Temminck & Schlegel, 1846) propagation using cryopreserved sperm samples

The aim of this study was to test the artificial seminal plasma (ASP) as the extender as well as methanol as the cryoprotectant for cryopreservation, and to collect information on the fertilizing capacity of cryopreserved sperm samples. Eggs from Japanese eel, Anguilla japonica, females were control‐fertilized with native sperm diluted with ASP and cryopreserved sperm (ASP as extender [in 1:100 ratio] and 10% methanol in v/v final concentration). No statistical differences (p < .05) were among the measured parameters (hatching, survival after 10 days post‐hatch, malformation rates) between the two groups, thus this cryopreservation method can be used effectively for artificial propagation of the Japanese eel, Anguilla japonica.     

牙鲆胚胎冷冻损伤的观察

为了观察牙鲆胚胎在冷冻保存过程中的形态受损情况,将其浸入20%PM（20%丙二醇和20%甲醇1∶1的混合液）中平衡,并用程序化法和玻璃化法对其冷冻保存2h后解冻,用摄影显微镜记录其在抗冻剂里平衡时和冷冻保存后的形态。结果显示在平衡过程中胚胎卵膜出现凹陷（称为溶液损伤）,但可以恢复;在冷冻过程出现胞内冰损伤,是致命的;用程序化法冷冻保存的尾芽期胚胎卵膜和卵黄完好,胚体边缘受伤;用玻璃化法保存的尾芽期胚胎,卵膜和卵黄损伤较重,胚体损伤较轻;因此将玻璃化法和程序化法结合可以达到扬长避短的效果。     

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 3β‐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.     

Sex differentiation and early gonadal development in Bombina orientalis (anura: Discoglossidae)

Gonadal differentiation in premetamorphic Bombina orientalis is described and staged. The pattern of events during differentiation in Bombina differs in several respects from that previously described in other anurans. The Bombina gonad initially develops on the ventral surface of the vena cava, where there is no pre-existent somatic genital ridge prior to the arrival of the germ cells. The sexually undifferentiated gonad does not have a distinct cortex and medulla; instead, medullary cells ingress from the mesonephric blastema during sexual differentiation. Formation of a testis or an ovary appears to depend on the ability (or lack of ability) of the medulla to invade the germ cell-containing cortex. In the germ line, sexual differetiation can be recognized by a premeiotic increase in oogonial cell volume.     

Sex steroid dynamics during embryogenesis and sexual differentiation in Eurasian perch, Perca fluviatilis

It is widely accepted that sex steroid hormones play an important and a specific role during the process of sex differentiation in fish. In order to describe the role of the three main sex steroid hormones (testosterone--T, 17beta-estradiol--E2 and 11keto-testosterone--11KT) during embryogenesis and sex differentiation in Eurasian perch, Perca fluviatilis, eggs, larvae and juveniles originating from two mixed-sex and two all-female progenies were regularly sampled from fertilization to hatching (D0) and from hatching to day 70 post-hatching (D70). Just after spawning, a significant amount of sex steroids [T (1634.2pgg(-1)), E2 (554.4pgg(-1)) and 11KT (1513.2pgg(-1))] was measured in non-fertilised eggs suggesting a maternal transmission of these steroids. From D2 to D70 post-hatching, E2 levels were significantly higher in mixed-sex progenies (median: 725.7pgg(-1)) than in all-female progenies (156.2pgg(-1)) and significantly increased after the onset of the histological differentiation of the gonad in both progenies (D35). Levels of 11KT were significantly higher in mixed-sex (median: 431.5pgg(-1)) than in all-female progenies (below the limit of assay detection) and significantly increased at D35 in all-female progenies (median value: 343.2pgg(-1)). Mean 11KT to E2 ratio was six-fold higher in mixed-sex progenies (1.35) than in all-female progenies (0.24). The data suggest that the 11-oxygenated androgen (11KT) plays a major role in the male differentiation process, and that sex differentiation in Eurasian perch is probably determined by the 11KT to E2 ratio.     

Modelling time‐varying low‐temperature‐induced mortality rates for pupae of Tuta absoluta (Gelechiidae,Lepidoptera)

In a series of laboratory experiments, acclimated pupae of Tuta absoluta were exposed to various constant low temperatures in order to estimate their maximum survival times (Kaplan–Meier, Lt_99.99). A Weibull function was fitted to the data points, describing maximum survival time as a function of temperature. In another experiment at ?6°C, the progress of mortality increasing with exposure time was identified. These values were fitted by a sigmoidal function converging asymptotically to 100% mortality for very long exposure times. Analysing mortality data from the maximum survival experiment by a generalized linear model showed a significant common slope parameter (p < .001) that reveals parallelism of the survival curves at each temperature if a log time axis is used. These curves appear stretched (time scaled) if plotted with a nonlogarithmic time axis. By combining these mathematical relations, it was possible to calculate a species‐specific ‘mortality surface’ which exhibits mortalities, depending on temperature and duration of exposure. In order to accumulate hourly mortalities for courses of varying temperatures, an algorithm was developed which yields mortality values from that surface taking into account the attained mortality level. In validation experiments, recorded mortalities were compared against modelled mortalities. Prediction of mortality was partially supported by the model, but pupae experiencing intensely fluctuating temperatures showed decreased mortality, probably caused by rapid cold hardening during exposure. Despite this observation, mortality data converged to distinct levels very close to 100% depending on the intensity of temperature fluctuations that were characteristic for different types of experiments. The highest mortality limit occurred at intensely fluctuating temperatures in laboratory experiments. This constituted a benchmark that was not reached under various field conditions. Thus, it was possible to identify temperature limits for the extinction of field populations of Tuta absoluta pupae.     

Characterization of Japanese flounder (Paralichthys olivaceus) NK-lysin, an antimicrobial peptide

The NK-lysin cDNA of Japanese flounder, Paralichthys olivaceus, consists of 657bp, containing an open reading frame (ORF) of 444bp, which encodes 147 amino acid residues. The amino acid sequence of Japanese flounder NK-lysin has 21% identity to porcine NK-lysin and bovine NK-lysin, 23% to equine NK-lysin, and 46% to zebrafish NK-lysin-like protein. Multiple alignments of Japanese flounder NK-lysin and other known saposin-like proteins revealed that the six cysteine residues important for structural folding are completely conserved. The Japanese flounder NK-lysin gene is approximately 2kb and consists of five exons and four introns. Japanese flounder NK-lysin mRNA constitutive expression was mainly detected in gills, heart, head kidney, intestines, peripheral blood leukocytes (PBLs), spleen and trunk kidney, and was detected at low levels in liver, muscle and ovary. However, expression was not detected in brain, skin and stomach of apparently healthy Japanese flounder. Gene expression of Japanese flounder NK-lysin was not inducible by lipopolysaccharide (LPS) treatment. A synthesized NK-lysin peptide, consisting of 27 amino acid residues, showed antimicrobial activity against Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Photobacterium damselae subsp. piscicida.     

Application of a temperature‐dependent fluorescent dye (Rhodamine B) to the measurement of radiofrequency radiation‐induced temperature changes in biological samples

We have applied a non‐contact method for studying the temperature changes produced by radiofrequency (RF) radiation specifically to small biological samples. A temperature‐dependent fluorescent dye, Rhodamine B, as imaged by laser scanning confocal microscopy (LSCM) was used to do this. The results were calibrated against real‐time temperature measurements from fiber optic probes, with a calibration factor of 3.4% intensity change °C^?1 and a reproducibility of ±6%. This non‐contact method provided two‐dimensional and three‐dimensional images of temperature change and distributions in biological samples, at a spatial resolution of a few micrometers and with an estimated absolute precision of around 1.5 °C, with a differential precision of 0.4 °C. Temperature rise within tissue was found to be non‐uniform. Estimates of specific absorption rate (SAR) from absorbed power measurements were greater than those estimated from rate of temperature rise, measured at 1 min intervals, probably because this interval is too long to permit accurate estimation of initial temperature rise following start of RF exposure. Future experiments will aim to explore this. Bioelectromagnetics 30:583–590, 2009. © 2009 Wiley‐Liss, Inc.     

Digestive capacity, growth and social stress in newly-metamorphosed Japanese flounder (Paralichthys olivaceus)

Hierarchies readily appear when rearing flounders through metamorphosis in space-limited conditions. In this experiment, subordinate fish were stressed, as suggested by their elevated cortisol level compared to dominant fish. Subordinate fish, although of smaller size than the dominant fish, showed no suppressed digestive capacity. By separating the two hierarchical groups into different tanks at low density, the cortisol level of the subordinate fish substantially decreased. The removal of the stressor also resulted in an increase in digestive function and improved coloration. Also, compensatory growth in length but not in weight was observed in the subordinate group, suggesting that the subordinate fish devoted their energy to increase their length. These results imply that the climax of flounder metamorphosis (i.e. the settlement stage) is a highly sensitive period, where social interactions may induce high levels of stress. However, Japanese flounder early juveniles prove to have a high recovery capacity from stress and show to allocate energy preferentially to growth in length. This seems to be an adaptation to diminish the probability of death by predation.     

Identification and differential expression of microRNAs during metamorphosis of the Japanese flounder (Paralichthys olivaceus)

Background

MicroRNAs (miRNAs) are a class of endogenous small non-coding RNAs of 20–25 nucleotides that play a key role in diverse biological processes. Japanese flounder undergo dramatic metamorphosis in their early development. The metamorphosis is characterized by morphological transformation from a bilaterally symmetrical to an asymmetrical body shape concomitant with extensive morphological and physiological remodeling of organs. So far, only a few miRNAs have been identified in fish and there are very few reports about the Japanese flounder miRNA.

Methodology/Principal Findings

Solexa sequencing technology was used to perform high throughput sequencing of the small RNA library from the metamorphic period of Japanese flounder. Subsequently, aligning these sequencing data with metazoan known miRNAs, we characterized 140 conserved miRNAs and 57 miRNA: miRNA* pairs from the small RNA library. Among these 57 miRNA: miRNA* pairs, twenty flounder miRNA precursors were amplified from genomic DNA. We also demonstrated evolutionary conservation of Japanese flounder miRNAs and miRNA* in the animal evolution process. Using miRNA microarrays, we identified 66 differentially expressed miRNAs at two metamorphic stages (17 and 29 days post hatching) of Japanese flounder. The results show that miRNAs might play a key role in regulating gene expression during Japanese flounder metamorphosis.

Conclusions/Significance

We identified a large number of miRNAs during flounder metamorphosis, some of which are differentially expressed at two different metamorphic stages. The study provides an opportunity for further understanding of miRNA function in the regulation of flounder metamorphosis and gives us clues for further studies of the mechanisms of metamorphosis in Japanese flounder.