Juvenile Japanese Flounder Research Articles

Toxic effects of combined exposure to cadmium and nitrate on intestinal morphology, immune response, and microbiota in juvenile Japanese flounder (Paralichthys olivaceus)

Cadmium (Cd2+) and nitrate (NO3−) are important environmental pollutants in the offshore marine ecological environment. However, limited research has explored their combined effects, particularly regarding their impact on the microbiota and intestinal health of marine fish. In this study, juvenile Japanese flounders (P. olivaceus) were immersed in seawater samples with different combinations of Cd2+ (0, 0.2, and 2 mg/L) and NO3− (0 and 80 mg/L NO3N) for 30 days to explore their toxic impacts on intestinal morphology, tight junction (TJ) barrier, immune response, and microbiota. Our results showed that Cd2+ or NO3− exposure alone led to histopathological damage of the gut, while their co-exposure aggravated intestinal damage. Moreover, co-exposure substantially decreased TJ-related gene expression, including occludin, claudin-10, and ZO-2, suggesting increased TJ permeability in the gut. Regarding the immune response, we observed upregulated expression of immune-related markers such as HSP40, IL-1β, TNF-α, and MT, suggesting the onset of intestinal inflammation. Furthermore, Cd2+ and NO3– exposure led to changes in intestinal microflora, characterized by decreased the abundance of Sediminibacterium and NS3a_marine_group while increasing the prevalence of pathogens or opportunistic pathogens such as Ralstonia, Proteus, and Staphylococcus. This alteration in microbiota composition increased network complexity and α-diversity, ultimately causing dysbiosis in the fish gut. Additionally, combined exposure resulted in metabolic disorders that affected the predicted functions of the intestinal microbiota. Overall, our study demonstrates that Cd2+–NO3– co-exposure amplifies the deleterious effects compared to single exposure. These findings enhance our understanding of the ecological risks posed by Cd2+–NO3– co-exposure in marine ecosystems.

Validation of otolith daily ring deposition and hatch date estimations in larval and juvenile Japanese flounder Paralichthys olivaceus

The early life history traits of Japanese flounder Paralichthys olivaceus, a commercially important species, have been well studied; however, daily ring formation in otoliths (lapilli and sagittae) of this species has not been investigated in detail. We examined the daily periodicity of ring deposition in otoliths using hatchery-reared Japanese flounder larvae and juveniles. In both lapilli and sagittae, a check corresponding to the radius at hatching (ca. 6 µm) was discernible throughout the pelagic larval and settled juvenile periods. The otolith rings were faint in the preflexion larval period and distinct in the flexion larval and later periods. Regression analyses for the number of rings by age in days showed that ring deposition occurred daily in lapilli but not in sagittae. Double marking of juveniles with alizarin complexone confirmed the daily ring periodicity in lapilli. However, the first faint ring was observed in the innermost areas in lapilli at 3–16 d post-hatching, and the faint rings were often unrecognisable, even under a scanning electron microscope, which interfered with daily age estimation. To estimate the number of days corresponding to the obscure zone with unrecognisable rings in lapilli, we developed the estimation method using logarithmic functions derived from the relationship between the radius (restricted to <20 µm) of lapilli and the ring counts for individual data. The error for the estimated days was − 0.05 ± 2.13 (n = 26, mean ± SD), indicating that this method is useful for hatch date estimation. Thus, the present study provides a novel method for analysis of otolith daily increments with unclear narrow rings in the innermost part of the otoliths.

Effect of dietary phosphorus level on defense system parameters and liver HSP70 concentration in juvenile Japanese flounder, Paralichthys olivaceus

Effect of dietary phosphorus level on defense system parameters and liver HSP70 concentration in juvenile Japanese flounder, Paralichthys olivaceus

Effects of feeding frequency on growth performances and nutrient digestibility of juvenile Japanese flounder, Paralichthys olivaceus (Temminck & Schlegel), under varied dietary protein levels

Effects of feeding frequency on growth performances and nutrient digestibility of juvenile Japanese flounder, Paralichthys olivaceus (Temminck & Schlegel), under varied dietary protein levels

Porous microplastics enhance polychlorinated biphenyls-induced thyroid disruption in juvenile Japanese flounder (Paralichthys olivaceus)

Microplastics and polychlorinated biphenyls are ubiquitous in the marine environments. To illuminate their combined biological impacts, juvenile Japanese flounder (Paralichthys olivaceus) were exposed to 500 ng/L PCBs alone or 500 ng/L PCBs plus 2, 20, and 200 μg/L 10-μm porous MPs for 21 days. Compared to PCBs alone, co-exposure to PCBs and 20, 200 μg/L MPs reduced fish body length and body weight, and the concurrence of MPs aggravated PCBs-induced thyroid-disrupting effects, including significantly decreased L-thyroxine and L-triiodothyronine levels, more severe damage to the thyroid tissue and gill morphology, and disturbance on the expression of hypothalamus-pituitary-thyroid axis genes. The PCBs concentrations in the seawater were decreased dramatically with the increase of MPs concentrations, confirming that MPs absorbed PCBs from the seawater. Our results demonstrated that MPs enhanced the thyroid disruption of PCBs, suggesting that the risk of MPs and thyroid-disrupting chemicals on marine organisms should be paid more attention.

Annual variation in the prey utilization of juvenile Japanese flounder in southwestern Hiuchi-Nada, Seto Inland Sea, Japan

Past studies indicated that newly settled juvenile Japanese flounder Paralichthys olivaceus depend on mysids as their main prey source, but little is known about the prey of juveniles in nursery grounds with a low density of mysids. This study aimed to reveal the annual variation in the main prey items of juvenile Japanese flounder in relation to their growth in southwestern Hiuchi-Nada, the Seto Inland Sea, Japan. Juvenile flounder consumed chiefly larval anchovy in 1995 and 1999, while small crangonid shrimp Crangon uritai were the main prey items in 1998, 2003, and 2004. The growth rates of juvenile flounder ranged from 0.82–1.33 mm d−1 during the five years of this study. Mysids were almost absent and small crangonid shrimp were abundant in the study area. Our results indicate that juvenile Japanese flounder prefer larval anchovy, but small crangonid shrimp function as the alternative prey when larval anchovy and mysids are not available in the study area.

Discrimination of nursery locations of juvenile Japanese flounder Paralichthys olivaceus on the Pacific coast of northern Japan based on carbon and nitrogen stable isotope ratios

To test whether stable isotope ratios of carbon (δ13C) and nitrogen (δ15N) can be used to understand the ecological connectivity of Japanese flounder Paralichthys olivaceus, we evaluated the isotopic signatures of juvenile P. olivaceus from the Pacific coast of northern Japan (i.e., from Aomori to Ibaraki Prefectures). We measured δ13C and δ15N in muscle and collagen extracted from vertebrae and found that isotope ratios were strongly correlated in both. The discrimination models we developed for muscle and collagen proved useful in distinguishing among juvenile P. olivaceus collected from the five prefectures (Aomori, Iwate, Miyagi, Fukushima, and Ibaraki). Juveniles collected in Aomori in particular showed significantly lower values of δ13C and δ15N than those from the other prefectures, which likely reflects the greater influence of the Tsugaru Warm Current in that region. Because collagen in the vertebral centrum can be used to reconstruct annual isotopic records, our results may also assist in developing migration tracking procedures for adult P. olivaceus along the Pacific coast of northern Japan.

Molecular adaptations of glucose and lipid metabolism to different levels of dietary carbohydrates in juvenile Japanese flounderParalichthys olivaceus

Japanese flounder juveniles (initial body weight: 7.12 ± 0.02 g) were fed three diets containing 0, 120 and 200 g/kg of carbohydrates (C0, C120 and C200, respectively) for 10 weeks. Results showed that higher dietary carbohydrate intake enabled further deposition of glycogen and lipids in liver and muscle. The mRNA levels of glucokinase (gck), phosphofructokinase (pfkl) and hexokinase (HK) activity involved in glycolysis were significantly up‐regulated in C120 (p .05) but significantly increased in C200 group (p < .05). In conclusion, Japanese flounder was able to use carbohydrates efficiently through regulation of glucose and lipid metabolism when dietary carbohydrate was not higher than 120 g/kg, while 200 g/kg of dietary carbohydrate caused the deregulation of glucose homoeostasis.

Skin darkening with increasing densities in juvenile Japanese flounder Paralichthys olivaceus

Skin darkening with increasing densities in juvenile Japanese flounder Paralichthys olivaceus

Cortisol promotes staining-type hypermelanosis in juvenile Japanese flounder

Hypermelanosis of the blind side of Japanese flounder frequently occurs in post-metamorphic juveniles reared in hatcheries. Due to the morphological similarity between darkened blind-side skin and normal ocular-side skin, hypermelanosis is regarded as a transition from normal blind-side skin to that of the ocular side. Hypermelanosis appears only in rearing tanks without sand on the bottom, and its incidence tends to increase under stressful conditions; however, the specific mechanisms responsible for its occurrence have not been identified to date. In teleosts, the hypothalamus-pituitary-interrenal axis (HPI axis) is activated under stress, which then accelerates cortisol secretion. Therefore, in this study, we orally administered cortisol to Japanese flounder to examine its effect on hypermelanosis. The results of our study indicated that cortisol promoted hypermelanosis in a dose-dependent manner. In addition, the effect of cortisol was canceled out in juveniles reared in tanks with bottom sand, suggesting that the well-documented inhibitory effect of bottom sand is stronger than the stimulatory effect of cortisol (i.e., stress) on hypermelanosis. However, administration of the cortisol antagonist RU486 failed to inhibit hypermelanosis, suggesting the contribution of other stimulatory factors to this process. Therefore, our findings strongly suggest that stress-induced cortisol release has the adverse effect of promoting hypermelanosis in juvenile flounders reared without bottom sand.

Evaluation of different Pyropia yezoensis extracts as feed additives for growth and immunity of Japanese flounder Paralichthys olivaceus

We evaluated the effects of diets incorporating the red algae Pyropia yezoensis, prepared by several different extraction methods, on the growth of juvenile Japanese flounder Paralichthys olivaceus. We assessed growth performance, as well as the levels of amino acids, fatty acids, insulin-like growth factor I (IGF-I) and interleukins (ILs). Four experimental diets were developed based on different methods of processing P. yezoensis. A commercial feed, laver powder (P), high-pressure heat extraction of laver (HPHE) and acid hydrolysis extraction of laver were used as the experimental diets. Three experimental replicates were established for each diet (40 fish/group, body weight 123.7 ± 1.1 g), and the fish were fed for 6 weeks. We found no significant differences in weight gain, specific growth rate or feeding efficiency among the groups (P > 0.05); however, the fish fed HPHE had the greatest growth performance. Fish fed the laver extracts exhibited the highest protein efficiency ratio compared with the control and P groups. The experimental groups fed P. yezoensis extracts had significantly higher levels of IGF-I (P < 0.05) than those of the control group. High levels of IL-2 were found in the P and HPHE groups, IL-12 in the HPHE group, and IL-6 in all experimental groups. Therefore, these results suggest that a P. yezoensis extract improved the growth performance and immunity of Japanese flounder. In particular, the high-pressure heating process was a useful extraction method for preparing a P. yezoensis extract, which had beneficial effects as a dietary supplement in Japanese flounder.

Survival, growth and physiological responses of juvenile Japanese flounder (Paralichthys olivaceus, Temminck &amp; 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.

Effect of sampling procedures on skin darkening in juvenile Japanese flounder Paralichthys olivaceus

Effect of sampling procedures on skin darkening in juvenile Japanese flounder Paralichthys olivaceus

Impact of the great tsunami in 2011 on the quality of nursery grounds for juvenile Japanese flounder Paralichthys olivaceus in Sendai Bay, Japan

AbstractThe impact of the great tsunami, which was generated by the Great East Japan Earthquake on 11 March 2011, on the quality of a nursery ground for juvenile Japanese flounder Paralichthys olivaceus with a sandy bottom of &lt;15 m in depth in Sendai Bay, Japan, was evaluated. Growth rates of juveniles, as an index of the quality of the nursery ground, were compared between before and after the tsunami. Recent growth rates (RGR; mm day−1) between 1 and 6 days before collection in 2011–2013, which were estimated from the width of otolith increments, ranged from 0.84 to 2.37 mm day−1 for juveniles of 24.8–146 mm in total length (TL). These figures are comparable to the maximum RGR for juvenile Japanese flounder in the literature. A linear model for RGR selected TL and bottom temperature but rejected biomass of the mysid Orientomysis mitsukurii, the main prey item, as explanatory variables, indicating that the growth rates of juveniles after the tsunami were not controlled by food density, i.e., optimal food condition occurred in the nursery. A linear model for observed growth rate (mm day−1), which was calculated from the temporal changes in the juvenile size frequency distribution, suggested the growth rate did not change between before and after the tsunami. Additional information on prey abundance and sediment size distribution also support this conclusion. Therefore, we conclude that the quality of the nursery ground for juvenile Japanese flounder in the study site has not been damaged by the great tsunami.

Ontogenetic changes in nucleic acid, protein contents, and growthof larval and juvenile Japanese flounder

In order to estimate relationships among growth potential and biochemical indicators of Japanese flounder, RNA and DNA as well as protein indices were measured from 5 days after hatching (DAH) to 45 DAH. Results showed that the RNA/DNA ratio and protein/DNA ratio had obvious relations with instantaneous growth rate (GM) and length-based instantaneous growth rate (GL) during the premetamorphic period. Significant negative correlations among protein/DNA ratio and GM and GL were then observed during metamorphosis. During the postmetamorphic phase, the RNA/DNA ratio was positively correlated with GM and GL. Data indicated that body growth of Japanese flounder is mostly hyperplastic before 20 DAH, hypertrophic until 27 DAH, and hyperplastic until the end. In order to investigate the effect of diel periodicity on RNA/DNA ratio in larvae of the fed and starved groups, an experiment was conducted for 2 days. Results showed that the average RNA/DNA ratio in the daytime was higher than that in the dark and the ratio in fed larvae was higher compared with that in starved ones. In order to examine starvation effect on RNA/DNA ratio, experiments were conducted from 20 DAH to 27 DAH. Juveniles were divided into five groups: a fed group and groups starved 1, 2, 3, and 4 days followed by refeeding. Results showed that the RNA/DNA ratio increased in the fed group and declined continuously in the starved group. After refeeding, the recovery of the RNA/DNA ratio was observed for 1-day, 2-day, and 3-day starved treatments but was not found for the 4-day starved treatment.

A simulation model for estimating optimum stocking density of cultured juvenile flounder Paralichthys olivaceus in relation to prey productivity

The determination of the optimum stocking density of hatchery-reared juveniles in relation to the carrying capacity of the nursery ground is important to the success of any stock enhancement programs. In order to estimate the surplus productivity of nursery grounds available to support hatchery-reared fish, a population growth model for juvenile Japanese flounder Paralichthys olivaceus was developed by extending an ecophysiology-based model on growth of juvenile fishes. Parameters of the model were adjusted using field-collected data from Ohno Bay in northeastern Japan. This model predicted the optimum stocking density, namely the maximum number of hatchery-reared juvenile Japanese flounder released into the bay without decreasing the growth of conspecific wild juveniles owing to food limitation. The optimum stocking density for juveniles of 89mm total length was estimated to be 2000 individuals per 250,000m2 in Ohno Bay in 1989. Under the model, the most important factors affecting the optimum stocking density of Japanese flounder were the abundance of mysids and wild juveniles at the time of release. The effect of abundance of a competing flatfish species on growth of wild Japanese flounder was less important.

Inter-annual variation in the surplus prey production for stocking of Japanese flounder Paralichthys olivaceus

Stocking hatchery fish into the wild should only be implemented at a level that can be sustained within the available food-based carrying capacity to avoid food resource competition with wild conspecifics. We constructed a simulation model to assess the growth of wild conspecifics under different magnitudes of hatchery fish releases, including no releases and actual release levels, and under different wild fish abundancies. Stocking of juvenile Japanese flounder Paralichthys olivaceus (approximately 70–110mm TL) in Fukushima from 2003 to 2005 was analyzed. We monitored post-release changes in abundance of both cultured and wild juveniles and their prey (mysids). Our survey and modelled simulations revealed that the surplus prey production was sufficient to support the consumption of stocked and wild Japanese flounder (up to 150,000 individuals per site in 2003 and 2004), during which wild juveniles achieved nearly maximum growth rates. However, in 2005, during which a dominant year class of wild Japanese flounder occurred, the body size of wild juveniles was 49% smaller than the predicted size based on the maximum growth rates. This suggests that the food-based carrying capacity was reached in that year. Thus, it is important to evaluate the existing prey and wild juveniles levels and adjust the release accordingly every year.

Enhancement of antibody response by LAMP1 chimeric antigen in a DNA vaccine

Immunization by an antigen-encoding DNA (DNA vaccines) has shown promising results in rendering protection against some bacterial and viral diseases in fish. However, some studies showed that DNA vaccines induce variable levels of protection compared with the other types of vaccination. These results are driving the research interest against these infectious diseases to improve the efficacy of DNA vaccines. Recently, chimeric antigens in DNA vaccines have been reported to increase the protection against certain diseases by enhancing specific antibody responses. In this study, we assessed the use of a chimeric DNA vaccine by using lysosomeassociated membrane protein-1 (LAMP1) fused with the major capsule protein (MCP) from red seabream iridovirus. First, Japanese flounder (Paralichthys olivaceus) LAMP-1 (jfLAMP1) gene was cloned and characterized through tissue distribution and expression analysis. The ORF for jfLAMP1 gene has a length of 1248 bp that encodes for 415 aa (about 44 KDa) and shows highly conserved transmembrane and cytoplasmic domains as those of higher vertebrates. Then, jfLAMP1 was used as a carrier of MCP gene by replacing the intra-lysosomal domain, and subsequent inserting into an expression vector, pCI-neo. In the vaccination trial, juveniles of Japanese flounder were distributed into four groups: pCMCP (MCP in pCI-neo vector), pCLAMP1-MCP (chimeric DNA of jfLAMP1 and MCP in pCI-neo), rMCP (recombinant MCP) and pCI-neo (empty vector as a control). At 30 days post vaccination, blood samples were taken and the serum antibody titers were measured by ELISA. The 3 vaccinated groups showed higher antibody titers than pCI-neo group. The pCLAMP1-MCP vaccinated fish showed antibody levels significantly higher than pCMCP (p<0.05). These results will serve as a basis for the use of LAMP1-chimeric DNA vaccines to increase the immune response against certain infectious diseases, for which conventional DNA vaccines show marginal efficacy.

有明海奥部および中部海域の近底層におけるアミ類の分布

Ariake Sound is a highly productive area and a good nursery for fish and shellfish species. Mysids are essential prey organisms for a number of juvenile fishes in Ariake Sound, but their distribution and abundance are not well understood. To investigate species composition and distribution, we collected hyperbenthic mysids with a sledge-net in the inner and central parts of Ariake Sound from 2001 to 2004. Fifteen species of mysid from nine genera were recorded. The numerically dominant species were Orientomysis tenuicauda, Erythrops minuta, Orientomysis aspera, Neomysis japonica, Rhopalophthalmus orientalis, and Hyperacanthomysis longirostris in order. Of these, O. tenuicauda and E. minuta were mainly collected at offshore stations with relatively high salinity. In contrast, the majority of O. aspera and the other three species were collected from nearshore stations and/or river estuaries. The highest density, 7,236 indiv./m2, was recorded for O. tenuicauda at a station with a sandy bottom at 20 m depth. Mean estimated density, 241 indiv./m2, was low compared with mysid densities recorded in other Japanese estuaries, but exceeded the density, 100 indiv./m2, known to maintain the maximum juvenile Japanese flounder growth rate in their nursery ground.

Dietary Soy Peptide Enhances Thermotolerance and Survival of Juvenile Japanese Flounder, Paralichthys olivaceus

AbstractSoy peptide (SP), a soy protein enzymatic hydrolysate, contains bioactive substances that could be utilized as an immune‐stimulating feed ingredient. The experiment evaluated the efficacy of dietary SP on promoting growth, and enhancing tolerance and survival to heat stress in juvenile Japanese flounder, Paralichthys olivaceus. Four diets were incorporated with different levels of SP (0, 2, 5, and 10%) and a 6‐wk feeding trial ensued. Following the feeding trial, the experimental groups were subjected to heat stress to measure survival rate and heat shock protein 70s (HSP70s) in gill, liver, and skin. Fish fed diets with SP inclusion showed considerable decrease in percent weight gain. Significantly higher lethal time values to 50% mortality (LT50) value were recorded for fish fed 10% SP. Moreover, LT50 values of fish fed 2 and 5% SP were significantly higher compared with fish fed control diet. HSP70s produced in all the tissues were significantly highest in fish fed 10% SP. HSP70s values were significantly higher in fish fed 2 and 5% SP compared with fish fed control diet. A significant reduction in HSP70s among all groups during recovery period was also observed. These results suggest that SP can be used to enhance the immune response and survival of P. olivaceus under heat stress.