Search

A pleuronectiform fish, the barfin flounder Verasper moseri, has three forms of gonadotropin-releasing hormone (GnRH) in the asymmetric brain: GnRH1 (seabream GnRH), GnRH2 (chicken GnRH-II), and GnRH3 (salmon GnRH). To determine whether the levels of GnRH differ between the ocular and blind sides of the forebrain of barfin flounder, we examined the distribution pattern of GnRH using time-resolved fluoroimmunoassays. Red seabream Pagrus major, which has a symmetric brain and three forms of GnRH, was used as a control. In contrast to red seabream, barfin flounder showed an imbalance in the weight of the forebrain (olfactory bulbs, telencephalon, and optic tectum-thalamus), with the ocular side (right side) significantly larger than the blind side (left side). In barfin flounder, there were significantly higher contents (pg/tissue) of the three forms of GnRH in the olfactory bulbs and optic tectum-thalamus in the ocular side of brain than in the blind side. In the telencephalon, GnRH2 content was significantly higher in the ocular side of brain. However, the contents of both GnRH1 and GnRH3 were not significantly different between the ocular and blind sides of the telencephalon, although those in the ocular side tended to be higher than those in the blind side. In red seabream, no difference in the contents of the three forms of GnRH was found between the right and left sides. Concentrations (pg/mg tissue) of the three forms of GnRH did not differ between the ocular and blind sides of the barfin flounder brain, as is the case of red seabream. These results suggest that in the barfin flounder, despite the ocular and blind sides of the forebrain differing in terms of brain volume, there are no functional or qualitative differences in the levels of the three GnRH forms because their tissue concentrations were the same on both sides.

We investigated movement pattern of commercial important fish around Offshore Wind Turbine (OWT) and neighboring habitats in Goto Islands. We tagged 55 individuals of 2 fish species and released around the OWT in 2017. (27 Pagrus major from 1st Feb 2017 to 16th Mar 2017 (winter), 8 P. major and 20 Seriola quinqueradiata from 13th July 2017 to 13th Oct 2017 (summer)). Acoustic receivers were deployed at OWT, south artificial reef (AS), trapnet (TN), north FAD (F1) and south FAD (F2) in winter and two more receivers were deployed at north artificial reef (AN) and natural reef (NR) in summer. We observed 12 P. major at OWT for a day after released in winter, majority of fish (13 individuals) were detected at TN. In summer, 7 P. major were observed at OWT, 1 visited TN within the day, but others disappeared. Ten S. quinqueradiata stayed at OWT longer than P. major after released and disappeared within 2 days. They mostly moved around AS, AN, NR. Residence time from K-M curve was 10 days for P. major in winter, a day in summer and 3 days for S. quinqueradiata. Both species showed low affinity for OWT as recorded low residency index despite the season.

The beneficial effects of Bacillus subtilis C-3102 and Saccharomyces cerevisiae type 1 were tested in red sea bream (Pagrus major) feeds. A basal diet (control) and two other diets were prepared by supplementation with B. subtilis C-3102 (PB) or S. cerevisiae type 1 (PY). After 60 days, both probiotic-supplemented groups exhibited significant enhancement in growth performance, the protein efficiency ratio (PER), and digestive enzyme secretion (protease and amylase) compared to the control group (PPPP<0.05). Catalase activity was also significantly decreased in both probiotic groups, with relatively lower activity observed in the PY group (P<0.05). Both probiotic groups showed considerably increased tolerance to freshwater exposure (P<0.05). In conclusion, B. subtilis C-3102 and S. cerevisiae type 1 can be used as functional probiotics to enhance the growth performance, digestion capacity, gut morphology, immune response, and stress resistance of the red sea bream with relatively higher efficiency by B. subtilis C-3102.

New two neutrophil types, sixth (N-6) and seventh type (N-7), were observed in the blood of Ceratothoa verrucosa (Cv)-infested (usual parasitism； N-6 and N-7 from two Cv-infested individual, N-7 from one Cv-infested individual) red seabream Pagrus major with exophthalmos. The bulging eyes contained bubbles. The N-6 and N-7 were observed in the infested fish without or with mouth insufficiency (MI； Mouth closure was incomplete), respectively； the exophthalmos occurred prior to MI (EP→MI).

Three types of granules, two extraordinary types and one inducible type, were observed in the neutrophils of red seabream Pagrus major infected with Tenacibaculum maritimum. Both extraordinary granule types (eoG1^{Tm}, eoG2^{Tm}) had similar morphologies but different cytochemical characteristics to those of ordinary granules (oG1^N, oG2^N) from non-infected fish. The eoG1^{Tm} showed chromophobic, simple morphology (without stratified structure), peroxidase positive and lack of lysozomal enzymes. The eoG2^{Tm} was stratified granule with three-layer structure [inner eosinophilic layer (L0-0), middle chromophobic layer (L0-1) and outer chromophobic layer (L1)]. Acid phosphatase, ꞵ-glucuronidase (ꞵ-Glu), α-naphtyl acetate esterase and naphthol AS-D chloroacetate esterase were detected in L0-0, but α-naphtyl butylate esterase was in L0 (L0-0 and L0-1). Peroxidase (PO) were localized in L1. Both extraordinary types were Sudan black B (SBB) negative. Spot formation, a curious phenomenon appeared in PO-stained oG2^N (positive L1 and negative L0), was not observed in eoG2^{Tm}. Inducible granule (iG^{Tm}) was consisted of two chromophobic layers (inner L0 and outer L1). The L0 of iG^{Tm} showed positive reaction to PO.

Appearance and behavioral abnormalities (AA and BA, respectively) of red seabream Pagrus major infested with Ceratothoa verrucosa (usual parasitism) were described in this report. Three major and some minor AA were observed in the infested fish [major： mouth insufficiency, exophthalmos and bubble formation in the eye (BF). minor： disappearance of bubble in the eye, bulge of cornea with bubble, rupture of the swollen cornea, storage of white turbid liquid in the eye]. On the other hand, BA were classified into five categories： 1. buoyancy dysregulation (BD； three types, a, b & c), 2. protruding action of the head above the water surface (PA), 3. shivering of the body, 4. sideways swing of the head and 5. body tilt. BD and PA were general, and others were rare. Exophthalmos, BF, BD and body tilt were speculated due to the dysregulation of blood gas concentration.

Red seabream Pagrus major cultured in our laboratory showed a symptom of cloudiness on the dorsal skin around the base of dorsal fin in June 2020. In the whitish skin, epithelial cells were enlarged with no proliferation. Mucus cells were not observed in the epithelium. Numerous lymphocytes accumulated in the dermis under the epithelium. Photobacterium damselae damselae was isolated from the whitish skin surface, however, the symptom (whitish skin) was not reproduced by experimental infection (immersion method) of this bacterium. Here, we call this disease dorsal skin cloudiness (DSC). Two types of granules were observed in the neutrophils of red seabream infected with DSC. Both granule types had similar morphologies but different cytochemical characteristics to those of ordinary granules (oG1^N, oG2^N) isolated from non-infected fish. In this paper, we called the two granule-types from the fish with the symptom of DSC as extraordinary chromophobic granules (type 1, eoG1^{DSC}： type 2, eoG2^{DSC}). The eoG1^{DSC} showed chromophobic, simple morphology (without stratified structure), peroxidase positive and lack of lysozomal enzymes. The eoG2^{DSC} was stratified granule with three-layer structure [inner eosinophilic layer (L0-0), middle chromophobic layer (L0-1) and outer chromophobic layer (L1)]. Lysozomal enzymes (acid phosphatase, β-glucuronidase and exterases ) and peroxidase (PO) were localized in L0-0 and L1, respectively. Both types of extraordinary granules were Sudan black B negative. Spot formation, a characteristic phenomenon seen in PO-stained oG2^N (positive L1 and negative L0), was not observed in eoG2^{DSC}.

To improve livestock and aquaculture-raised fish as food, targeted mutagenesis using genome editing technologies is becoming more realizable. Myostatin (Mstn), which functions as the negative regulator of skeletal muscle growth, is one of the major targets to improve the edible ratio of livestock and farmed fish. We previously reported that the deficiency of Pm-mstn, one of Myostatin paralogs, improves muscle growth and changes body shape in a finfish species, red seabream (Pagrus major), as a result of editing the gene by means of CRISPR/Cas9. In this study, we established Pm-mstnb-deficient red seabream, which is a null-allelic mutant of another paralogous gene of Myostatin in the species, and analyzed their phenotype in terms of growth traits and body shape. A comparison of all growth traits between Pm-mstnbwt/wt and Pm-mstnb-5/-5 revealed no significant differences. In addition, all metrics for body shape, defined as the ratios of body depth, body width, and depth of the caudal peduncle to body length, respectively, were also similar in Pm-mstnbwt/wt and Pm-mstnb-5/-5. Therefore, we concluded that Pm-mstnb does not function as a negative regulator of skeletal muscle growth in red seabream.

A formalin-inactivated red sea bream iridovirus (RSIV) vaccine was prepared using the culture supernatant of a persistently infected Pagrus major fin cell line (PI-PMF) with IVS-1 strain (RSIV subtype II Meglaocytivirus). Rock bream (Oplegnathus fasciatus) were injected with a high-dose, ultracentrifuged megalocytivirus vaccine (Ultra HSCMV, 7.0 × 1010 copies/mL), a high-dose supernatant of cultured megalocytivirus vaccine (HSCMV, 1.0 × 1010 copies/mL), a supernatant of cultured megalocytivirus vaccine (SCMV, 1.0 × 109 copies/mL), and a low-dose of cultured megalocytivirus vaccine (LSCMV, 1.0 × 108 copies/mL). The vaccine efficacies for the various vaccine formulations were determined done following injection challenge with IVS-1 (1.0 × 104 copies/0.1 mL/fish), and the four different vaccines exhibited cumulative mortalities of 10.0 ± 0.0%, 48.3 ± 7.6%, 75.0 ± 5.0%, and 100.0 ± 0.0%, respectively. Additionally, the dose-dependent vaccine efficacy was also confirmed using two different cohabitation methods that included challenges G (general) and I (individual). When squalene + aluminum hydroxide (SqAl) was used as an adjuvant for the HSCMV or SCMV vaccine, cumulative mortalities of 30.0 ± 5.0% and 48.3 ± 7.6%, respectively, were obtained; moreover, these two adjuvants exhibited the highest efficacy in this study. The observed difference in survival post-challenge for the different vaccine concentrations was not reflected in the differences in neutralizing antibody titers. It was found that the water temperature during immune induction plays a less important a role than the water temperature during the challenge test, in which lowering the water temperature from 25 °C to 21 °C during a challenge improved the level of protection from cumulative mortalities from 35% to 10%. This study demonstrated that protection against mortality using inactivated vaccines against RSIVD in rock bream, which are known to be the most susceptible species to RSIV infection, is dependent upon antigen dose and temperature during the challenge.

We evaluated the efficacy of mozuku fucoidan supplementation to alternative dietary proteins used in fish meal (FM) replacement to enhance growth, immunity, and stress resistance of Pagrus major. Seven isonitrogenous (45% protein) experimental diets were formulated where diet 1 (D1) was FM-based control diet. Diets 2 to 7 were formulated by replacing 25, 50, and 75% of FM protein with soy protein isolate (SPI) protein, and each replacement level was supplemented without or with fucoidan at 0.4% for diet groups D2 (FM25), D3 (FM25Fu), D4 (FM50), D5 (FM50Fu), D6 (FM75), and D7(FM75Fu), respectively. Each diet was randomly allocated to triplicate groups of fish (4.1 g) for 56 days. Significantly higher weight gain and specific growth rate were observed in fish fed FM50Fu diet group, and it was not differed (P > 0.05) with fish fed FM25Fu diet group. FM-based control diet showed intermediate value, and it was not differed (P > 0.05) with or without fucoidan-supplemented ≤ 50% FM replacement groups and FM75Fu diet group. Significantly lower growth performances were observed in FM75 diet group. At each replacement level, fucoidan-supplemented groups showed nonsignificant improvement of feed utilization performances. Fish fed fucoidan-supplemented diets showed best condition of oxidative and freshwater stress resistance. Lysozyme activity, NBT, and peroxidase activity showed higher (P > 0.05) values in fucoidan-supplemented groups compared with the non-supplemented groups. Catalase activity was significantly lower in FM75Fu diet group. Catalase activity is significantly influenced by the interaction effects of fucoidan and FM replacement level. In conclusion, fucoidan supplementation could increase the efficiency of utilizing SPI (≥ 75%) without any adverse effects on red sea bream performance.

Cholecystokinin (Cck) and peptide YY (Pyy) play important roles in digestive regulation in fish, but their function in red seabream (Pagrus major) remains obscure. Two cck complementary DNAs (cck-1 and cck-2) in red seabream were identified, and tissue distribution analysis of the genes revealed that cck and pyy were expressed throughout the digestive tract, except for stomach cck-2. After feeding, cck-1 expression significantly increased in the stomach and pyloric caeca, cck-2 expression significantly increased in the pyloric caeca and hindgut, and pyy expression increased in the pyloric caeca of red seabream. After oral administration of dietary protein, i.e., high-quality fish meal (HQFM), soy protein concentrate (SPC), or low-quality FM (LQFM), only HQFM significantly increased stomach cck-1 expression. To assess the growth of red seabream in response to different sources of dietary protein, three isonitrogenous and isoenergetic diets were formulated: HQFM, SPC with a low percentage of HQFM, and LQFM. Mean body weight was highest in the HQFM group after 6 weeks. The stomach cck-1 response to HQFM may explain the high growth in the HQFM group. Our data suggest that cck-1, cck-2, and pyy are involved in digestion. Furthermore, stomach cck-1 may be a valuable tool for evaluating the quality of dietary protein sources for red seabream.

The practical difficulty of parenteral application of fish vaccines against devastating fish diseases diverted the interest toward oral vaccination. Search for effective methods to enhance the oral uptake of viral and bacterial vaccines is continuing. The current research focus on a new role of mucosal fish vaccine adjuvants inducing the antigen uptake by enhancing vascularity or increasing intestinal permeability. Some inflammatory substances cause reversible pathology to the intestinal epithelium, which could be employed for the transepithelial passage of vaccine particles. The natural inflammatory substances used were capsaicin, piperine, and okadaic acid as 1 mg, 2 mg, and 1 μg/fish, respectively. Two inactivated vaccines were used as antigens to test the effect of these inflammatory substances in two different fish hosts. Tested vaccines were inactivated redspotted grouper nervous necrosis virus vaccine in sevenband grouper (Epinephelus septemfasciatus) and inactivated Edwardsiella tarda vaccine in red sea bream (Pagrus major) fish models. The inflammatory substances and each vaccine were anally intubated to fish. Capsaicin proved to be effectively aiding the transepithelial passage of vaccine particles more than piperine, while okadaic acid had no detectable effect.

Calpains (CAPNs) belong to the papain superfamily of cysteine proteases, and they are calcium-dependent cytoplasmic cysteine proteases that regulate a variety of physiological processes. We obtained the sequence of CAPN3 from an NGS-based analysis of Pagrus major (PmCAPN3) and confirmed the conserved molecular biological properties in the predicted amino acid sequence. The amino acid sequence and predicted domains of CAPN3 were found to be highly conserved in all of the examined species, and one catalytic domain and four calcium binding sites were identified. In healthy P. major, the PmCAPN3 mRNA was most abundantly expressed in the muscle and skin, and ubiquitously expressed in the other tissues used in the experiment. After artificial infections with fish pathogens, significant changes in its expression levels were found in immune-related tissues, most of showed upregulation. In particular, the highest level of expression was found in the liver, a tissue associated with protease activity. Taken together, these results suggest a physiological activity for PmCAPN3 in P. major and reveal functional possibilities that have not yet been reported in the immune system.

We report the morphology and cytochemistry of new neutrophil types (fourth type, N-4^th； fifth type, N-5^th) from Ceratothoa verrucosa (Cv)- infested red seabream Pagrus major with morphological abnormalities. The number of Cv in each infested fish was one (usual parasitism). The N-4^th and N-5^th were observed in the infested fish with mouth insufficiency (MI； Mouth closure was incomplete) and with MI plus exophthalmos (ocular proptosis), respectively. The bulging eyes contained bubbles. The first (N-1^st), second (N-2^nd) and third (N-3^rd) neutrophil types have been reported from Cv-infested fish showing usual parasitism without morphological abnormality, unusual parasitism with MI, and super-unusual parasitism with MI plus exophthalmos, respectively. Therefore, the N-4^th and N-5^th will be the intermediate types (N-4^th, between N-1^st and N-2^nd； N-5^th, between N-1^st and N-3^rd).

Three types of granules, two extraordinary types and one inducible type, were observed in the neutrophils of red seabream Pagrus major infected with Vibrio harveyi. Both extraordinary granule types (eoG-1^Vh, eoG-2^Vh) had similar morphologies but different cytochemical characteristics to those of ordinary granules (oG-1, oG-2) from non-infected fish. The eoG-1^Vh showed chromophobic, simple morphology (without stratified structure), peroxidase positive and lack of lysozomal enzymes. The eoG-2^Vh was stratified granule with twolayer structure [inner eosinophilic layer (L0) and outer chromophobic layer (L1)]. Lysozomal enzymes [acid phosphatase, ϐ-glucuronidase (ϐ-Glu) and esterases] and peroxidase (PO) were localized in L0 and L1, respectively. Both extraordinary types were Sudan black B (SBB) negative. Spot formation, a curious phenomenon appeared in PO- stained oG-2 (positive L1 and negative L0), was not observed in eoG-2^Vh. Many L0 of eoϐG-2^Vh were ϐ-Glu positive. Contrastively, this enzyme activity was detected in a few L0 of oG-2. Inducible granule (iG^Vh) was consisted of two chromophobic layers (inner L0 and outer L1). The L0 of iG^Vh showed positive reaction to alkaline phosphatase, PO and SBB. Esterases were detected not only in the L0 of eoG-2^Vh but also in the L1 of iG^Vh.

BackgroundHenneguyaThélohan, 1892 (Myxobolidae) is one of the most species-rich genera of myxosporean parasites infecting fish. Although common in nature, there are few reports of these parasites causing important disease in aquaculture. In this paper, we describe a new species ofHenneguyainfectingPagrus major(Temminck & Schlegel), a fish host introduced to the Mediterranean Sea from Japan in the late 1980s.ResultsLarge plasmodia of the parasite were found in the bulbus arteriosus and in the ventricle of the infected fish. Spores were found mainly in the kidney and heart and were accompanied by melanized macrophages or vascular intimal proliferation mixed with a mild non-suppurative response, respectively. Comparisons of morphometric data for spore and polar capsule length and width, suggest a unique combination of features in the newly described species. Molecular analysis, based on18SrDNA sequence of the parasite, followed by phylogenetic analysis, indicated that the parasite described here is a novel species ofHenneguya, clustered with the marine congeneric species.ConclusionsHenneguya aegean. sp. infects in aquacultureP. major, a host introduced as eggs to the Mediterranean from Japan. Despite the high host specificity of the myxobolid parasites,H. aegean. sp. seems to be able to useP. majoras a host and propagate successfully, causing morbidity and mortality. This could result in spillback of the new species from high density cultured non-nativeP. majorto native fish hosts.

Red sea bream (Pagrus major) is described as one of the marine fishes with high economic value, which is widely cultivated in East Asia. With large-scale and intensive aquaculture, bacterial diseases outbreak more frequently than before. In order to effectively prevent and control Vibrio anguillarum disease in red sea bream, we systematically analyzed the immune response mechanism of the head kidney and spleen of P. major infected with Vibrio anguillarum by RNAseq technology. In this study, we obtained 225,106,036 clean reads. By using Nr, Nt, Pfam, KOG/COG, Swiss-prot, KEGG, GO databases, we annotated 85,595 genes. In the head kidney and spleen, we obtained 3963 and 4981 DEGs (different expressed genes) respectively. To further study the function of DEGs, we performed GO and KEGG enrichment analysis on them. In pathway enrichment, the pathways which significantly enriched in both head kidney and spleen included Antigen processing and presentation, Phagocytosis and Cell adhesion molecules. In the head kidney, Ubiquitin-mediated proteolysis was specially enriched; while Intestinal immune network for IgM production, Complement and coagulation cascade were specially enriched in the spleen. This study provide effective data support for the comprehensive analysis of mechanism in P. major immune response following V. anguillarum infection, which would lay the foundation for further disease prevention and treatment.

A 60-day experiment was designed to assess the effect of different ratios of fish meal (FM): camelina meal plant protein (CM) on growth response and relative gene expression of growth-promoting factors, feed utilization potency, digestive enzymes activities, apparent digestibility (ADC), stress response, non-specific immunity of Pagrus major. Four isonitrogenous (490.7 g/kg of crude protein) and isolipidic (91.5 g/kg total lipid) experimental diets were formulated and designated as camelina meal (CM0), soyabean meal (SBM20.5), CM20.5, and CM33 based on protein contents. At the end of the feed trial, significantly higher (p &lt, 0.05) weight gain, specific growth rate, and feed intake but lower feed conversion ratio were recorded in fish fed CM0, SBM20.5, and CM20.5 than fish fed CM33. The lowest growth, feed utilization, enzyme activity, and digestibility were recorded in fish fed CM33. Significantly higher pepsin, amylase, and protease activities were observed in fish fed CM0, SBM20.5, and CM20.5 diets than fish fed CM33. The highest ADC of protein was recorded in fish fed CM0, SBM20.5, and CM20.5 diets. Hematocrit levels were depressed CM33 while total serum protein, total cholesterol, triglyceride, blood urea nitrogen, total bilirubin, aspartate aminotransferase, and alanine aminotransferase were not significantly changed by the inclusion of CM. Non-specific immune variables (lysozyme activity, peroxidase activity in serum and nitro blue tetrazolium) in fish fed CM0, SBM20.5, and CM20.5 were significantly higher than in fish fed CM33 diet. The superoxide dismutase of fish fed CM20.5 was not significantly different from CM0 and SBM20.5 (p &gt, 0.05). Catalase and low salinity stress test show that CM0, SBM20.5, and CM20.5 were not significantly (p &gt, 0.05) different, while CM33 was significantly lower than the rest of the diets. TBARs show that CM20.5 and CM33 diets were significantly different (p &lt, 0.05), but CM20.5 was not significantly different from SBM20.5. Significantly higher hepatic IGF-1 and IGF-2 mRNA expression was found in fish-fed diet groups CM0, SBM20.5, and CM20.5 than fish fed CM33. The present study indicated that the addition of CM up 205 kg/kg to diet maintains growth, digestive enzymes, nutrient digestibility, immunity, stress resistance, and feed utilization efficiency of red sea bream.

Red sea bream iridoviral disease (RSIVD) causes high economic damage in mariculture in Asian countries. However, there is little information on the source of infection and viral dynamics in fish farms. In the present study, the dynamics of RSIV in a fish farm that mainly reared juveniles and broodstocks of red sea bream (Pagrus major) were monitored over 3 years (2016 to 2018) by targeting environmental DNA (eDNA) of seawater. Our monitoring demonstrated that red sea bream iridovirus (RSIV) was detected from the eDNA at least 5 days before an RSIVD outbreak in the juveniles. The viral loads of eDNA during the outbreak were highly associated with the numbers for daily mortality, and they reached a peak of 106 copies/liter seawater in late July in 2017, when daily mortality exceeded 20,000 fish. In contrast, neither clinical signs nor mortality was observed in the broodstocks during the monitoring periods, whereas the broodstocks were confirmed to be virus carriers by an inspection in October 2017. Interestingly, the viral load of eDNA in the broodstock net pens (105 copies/liter seawater) was higher than that in the juvenile net pens (104 copies/liter seawater) just before the RSIVD outbreak in late June 2017. After elimination of all RSIV-infected surviving juveniles and 90% of broodstocks, few RSIV copies were detected in the eDNA in the fish farm from April 2018 onward (fewer than 102 copies/liter seawater). These results imply that the virus shed from the asymptomatically RSIV-infected broodstock was transmitted horizontally to the juveniles and caused further RSIVD outbreaks in the fish farm. IMPORTANCE Environmental DNA (eDNA) could be applied in monitoring waterborne viruses of aquatic animals. However, there are few data for practical application of eDNA in fish farms for the control of disease outbreaks. The results of our field research over 3 years targeting eDNA in a red sea bream (Pagrus major) fish farm implied that red sea bream iridoviral disease (RSIVD) outbreaks in juveniles originated from virus shedding from asymptomatically virus-infected broodstocks. Our work identifies an infection source of RSIVD in a fish farm via eDNA monitoring, and it could be applied as a tool for application in aquaculture to control fish diseases.

Infectious hematopoietic necrosis virus (IHNV) is known as a causative agent of heavy mortalities in farmed rainbow trout. However, there is limited information on its virulence for marine fish species. In this study, Japanese amberjack Seriola quinqueradiata and red sea bream Pagrus major were experimentally infected by intraperitoneal (IP) injection and immersion, with an IHNV isolate from rainbow trout in Japan, to evaluate the virulence of the virus for these fish species. The cumulative mortality for immersed rainbow trout was 15%. IHNV was isolated from all dead fish and 50% of the sequentially sampled rainbow trout. When Japanese amberjack were challenged by IP injection and immersion, the resulting cumulative mortality was 70% and 0%, respectively. The virus was isolated from all dead fish and 1 out of 3 Japanese amberjack sampled at 9 d post exposure. However, no mortality was observed in either of the red sea bream groups challenged with IHNV. IHNV was not isolated from any of the surviving red sea bream, or from any of the sequentially sampled fish. The viral titer on Japanese amberjack-derived YTF cells was in the same log range as that on FHM and RTH-149 cells, but the titers on the red sea bream cell lines SBK and GBRS were lower than the other cell lines, and were significantly different from the FHM and RTH-149 cell lines. These results suggest that Japanese amberjack has a low susceptibility to IHNV, and red sea bream has no or little susceptibility to the virus.

A 56-day feeding trial to evaluate the responses of red seabream (initial weight: 1.8 ± 0.02 g) to the substitution of fish oil (FO) with camelina oil (CO) at different ratios was conducted. The control diet formulated at 46% CP (6F0C) contained only FO without CO, from the second to the fifth diet, the FO was substituted with CO at rates of 5:1 (5F1C), 4:2 (4F2C), 3:3 (3F3C), 2:4 (2F4C), and 0:6 (0F6C). The results of the present study showed that up to full substitution of FO with CO showed no significant effect on growth variables BW = 26.2 g–28.3 g), body weight gain (BWG = 1275.5–1365.3%), specific growth rate (SGR = 4.6–4.7), feed intake (FI = 25.6–27.8), feed conversion ratio (FCR = 1.0–1.1), biometric indices condition factor (CF = 2.2–2.4), hepatosomatic index (HSI = 0.9–1.1), viscerasomatic index (VSI = 7.5–9.5), and survival rates (SR = 82.2–100) with different FO substitution levels with CO. Similarly, there were no significant differences (p &lt, 0.05) found in the whole-body composition except for the crude lipid content, and the highest value was observed in the control group (291 g/kg) compared to the other groups FO5CO1 (232 k/kg), FO4CO2 (212 g/kg), FO2CO4 (232 g/kg) and FO0CO6 (244 g/kg). Blood chemistry levels were not influenced in response to test diets: hematocrit (36–33%), glucose (Glu = 78.3–71.3 mg/dL), total protein (T-pro = 3.1–3.8 g/dL), total cholesterol (T-Chol = 196.0–241 mg/dL), blood urea nitrogen (BUN = 9.0–14.6 mg/dL), total bilirubin (T-Bil = 0.4–0.5 mg/dL), triglyceride (TG = 393.3–497.6 mg/dL), alanine aminotransferase test (ALT = 50–65.5 UL/L), aspartate aminotransferase test (AST = 38–69.3 UL/L). A remarkable modulation was observed in catalase (CAT) and superoxide dismutase (SOD) activities in the liver, as CAT and SOD values were lower with the complete FO substitution with CO (0F6C), and the highest values were observed in the control and (4F2C). This study indicates that red seabream may have the ability to maintain LC-PUFAs between tissues and diets, and CO substitution of FO could improve both lipid metabolism and oxidation resistance as well as maintain digestibility. In conclusion, dietary FO can be replaced up to 100% or 95% by CO in the diets of red seabream as long as n-3 HUFA, EPA, and DHA are incorporated at the recommended level.

Heavy rainfall can reduce salinity and increase turbidity in coastal waters, creating stressful conditions for the organisms found there, especially for the early stages of their ontogeny. The effects of salinity and turbidity on the embryonic stages of red sea bream Pagrus major were examined under controlled laboratory conditions. In the first experiment, eggs of red sea bream were exposed to different salinity stresses [34 (control), 30, 26, 22, 18, 14 psu] for 3 h. In the second experiment, eggs were exposed to higher turbidity levels of 100, 300, 500, and 700 NTU from 0 NTU (control) obtained by dissolving kaolin clay for the same duration as in the first experiment. The results showed that the embryos of red sea bream are tolerant to short-term exposure to low salinity stress. There were no significant differences in the hatching rate, yolk sac consumption of newly hatched larvae, or survival of larvae at 6 days post-hatching. However, the short exposure to turbidity stress reduced the hatching rate, total length, and yolk sac volume of newly hatched larvae, and larval viability of red sea bream. Turbidity had a significant impact on the abnormality rate at hatching, with higher values observed for elevated turbidity.

In aquaculture, periodic measurement of fish body size is required to suitably assess growth progress. The aim of this study is to monitor the growth of free-swimming red sea bream, Pagrus major, farmed in net cages using multi-stereo-image measurement to determine the efficacy and potential applications of the method. After estimating appropriate sample sizes based on mean fork length and varying acceptable error ranges, we tracked fish growth using both multi-stereo-image measurement and traditional scoop-net capture methods for 1 year. Measurements were conducted four times every 3 months. In fish with fork length exceeding 40 cm, the appropriate sample sizes with acceptable error ranges of 1%, 2%, 3%, 4%, and 5% were around 100, 26, 12, 7, and 5 individuals, respectively, based on a 95% confidence interval. The fish exhibited most of their growth from June to December, with slower growth from February to June. This study provides evidence that multi-stereo-image measurement is an accurate and useful tool to enhance understanding of growth processes in cultured fish.

Euryhaline rotifer belonging to the genera Brachionus are often used as live feed for first feeding marine finfish larviculture. Two common rotifer enrichments, a single usage of docosahexaenoic acid (DHA)-enriched Chlorella vulgaris and a combination of DHA-enriched C. vulgaris and salmon roe emulsion oil were used to assess: (i) nutritional status of rotifers and, (ii) performance when rotifers were fed to Pagrus major and Paralichthys olivaceus larvae from 2 to 15 days after hatching (DAH). Both rotifer enrichment treatments were found to be nutritionally effective with high unsaturated fatty acid (HUFA) content, especially in the combined treatment with significantly higher eicosapentaenoic acid (EPA). Despite deficient levels of EPA, DHA-enriched C. vulgaris was found to be a suitable enrichment for rotifer and also had positive response in survival, growth and trypsin activity in both P. major and P. olivaceus. DHA-enriched C. vulgaris only treatment had higher rotifer population growth (13% higher), egg bearing capacity (7% higher), almost double the soluble protein content and generally higher free amino acid content. Oxidation of lipids in the combined diet due to its high-saturated fatty acid content could be a possible cause to the inferior performance of the combined treatment. Both P. major (20.1 ± 10.1%) and P. olivaceus (12.3 ± 3.87%) suffered low survival and lower gut content in the combined treatment especially during periods of significant growth in both species (7–11 DAH). Despite high HUFA content in the combined treatment, this study revealed that consideration of free amino acids and soluble protein content as parameters of nutritional indicators for first feeding P. major and P. olivaceus should not be discounted.

A feeding experiment was conducted to develop non‐fish meal and non‐fish oil diet for red seabream by using plant protein source and Schizochytrium meal which is rich in 22:6n‐3 (DHA). Three iso‐nitrogenous and iso‐lipidic experimental diets were prepared (CP 41.2% ± 0.4%, CL 16.4% ± 1%). Control diet contained both fish meal (40%) and fish oil (6%). In the second diet, fish meal was replaced by plant meals (soy protein concentrate, soybean meal, corn gluten meal) [FO]. In the third diet, fish meal and fish oil were replaced by algae meal (Schizochytrium sp. powder) and plant proteins [AO]. Duplicated groups of juvenile red seabream (8.8 g ± 1.5) were fed the experimental diets for 12 weeks to near satiation. There was no statistical difference among treatment in specific growth rate. Feed conversion ratio of AO diet group was higher than that of control. In wet basis, whole body protein level was significantly higher in AO diet than FO group while lipid content was lower than control group. In fatty acid profile, AO group had significantly lower 18:4n‐3, 20:4n‐3, 22:5n‐3 and 20:5n‐3 (EPA) level, but significantly higher 18:3n‐3 and DHA level than the other two diet fed fish. The results might suggest that further developments in microalgae diet offer a promising lipid source of n‐3 PUFA as essential fatty acid on marine fish. And it showed possibility to develop non‐fish meal and non‐fish oil feed for marine aquaculture fish by using microalgae.

Tetraploid fish are important for mass production of triploids in polyploid breeding. In this study, we reported a novel protocol for artificial induction of tetraploidy by a combination of cold shock and hydrostatic pressure administered to gynogenetically developed eggs in Japanese flounder Paralichthys olivaceus. The induction was carried out by activating the eggs with UV-irradiated sperm of red sea bream Pagrus major, administering a cold shock (0°C, 5 to 45 min) 3 min after fertilization to inhibit second polar body exclusion, incubating the eggs for 60 min at 17°C, and treating them with a 650 kg/cm2 hydrostatics pressure shock for 6 min. We named the embryos gyno-tetraploids that developed from eggs after such treatments. The hatching rate of the gyno-tetraploids ranged from 20.99%±3.66% to 36.01%±2.79%, and the tetraploid rate ranged from 80.00% to 100.00%. All-maternal inheritance was verified using 6 high-recombination-rate microsatellite markers. This method successfully induced gyno-tetraploidy. The successful induction of gyno-tetraploidy lays the foundation for triploidization of new varieties with improved economic traits of interest that can benefit commercial culture.

Habitat preference is thought to be genetically programmed in fishes. However, fishes can choose habitat based on their personal experience of an environment. We investigated whether the environment in which fish are raised affects habitat preference in red sea bream Pagrus major juveniles, and tested if the formed preference lasts until later life stages. Juveniles were reared in tanks with a substrate of either sand or artificial seaweed for 40 days. Naive fish were raised without either type of substrate. In the preference test, individual fish were allowed to choose either a sand or artificial seaweed microhabitat. The tested fish were then kept in barren tanks, and similar tests conducted again on days 30 and 100. Sand and seaweed treatment fish preferred the corresponding habitat immediately after the rearing treatment, whereas naive fish did not exhibit any preference. These preferences were maintained when fish were tested on day 30, but not on day 100. The present study suggests that habitat preference is acquired through the rearing environment at the nursery stage, and that this preference lasts for at least 30 days. The formation of habitat preference should help juveniles to choose an optimal microhabitat in a fluctuating environment.

Two trials were carried out to determine the optimal replacement level of fish meal (FM) by soy protein concentrate (SPC) derived from soymilk without supplementation of indispensable amino acids (IAAs) or palatability enhancers and whether phytase supplementation can help to replace more SPC in diet of juvenile red sea bream, Pagrus major. In Trial 1, five isoenergetic diets were formulated: 67% FM as protein source in the control diet (C), and FM was replaced by 60 (SPC60), 70 (SPC70), 80 (SPC80) and 100% (SPC100) by SPC. In Trial 2, diets C and SPC80 were the same as in Trial 1, and phytase was supplemented in SPC80 at 1000 (P1), 2000 (P2), 3000 (P3) and 4000 (P4) FTU/kg diet. Thirty (ca. 23 g) and 20 fish (ca. 21 g) were randomly distributed into each fifteen and eighteen 300 L indoor tanks in Trial 1 and 2, respectively. Fish in both trials were fed two times daily until apparent satiation for 10 weeks. At the end of both trials, feces were collected after feeding with chromic oxide (Cr2O3) mixed diets. In Trial 1, there were no significant differences in final mean weight, specific growth rate (SGR), daily feeding rate (DFR), feed efficiency (FE), condition factor (CF), and retention efficiency of protein, lipid and energy until 70% (SPC70) FM replacement by SPC (P > .05). However, 80% FM replacement significantly reduced final mean weight, FE and lipid retention efficiency compared with group C (P .05). Final mean weight, FE, P digestibility, and retention efficiency of protein, lipid and P were significantly improved in fish fed diet P2 (phytase at 2000 FTU/kg diet) compared with those fed diet SPC80. However, phytase supplementation did not stimulate the growth up to the level of group C. The results demonstrated in juvenile red sea bream that 70% of FM can be replaced by SPC derived from soymilk without supplementation of IAAs and palatability enhancers.

We developed a novel strain of red sea bream Pagrus major that showed resistance to red sea bream iridoviral disease (RSIVD) using marker-assisted selection combined with DNA-based family selection and evaluated survival of its G2 in a farm environment. The G1 population was derived from a single RSIVD-resistant male (G0). The G1 progeny of the resistant male were identified by DNA parentage analysis using microsatellites. Marker-assisted selection using a quantitative trait locus linked to the RSIVD-resistant trait (Pma4_014) was conducted, and individuals inheriting the resistant allele of Pma4_014 (+) were selected. Two G2 populations were produced in 2016 and 2017, and reared for approximately three months, during the summer, at an aquaculture facility. A normal production cohort was also reared at the same farm in 2016 and survival compared. Survival rates of the G2 populations produced in 2016 and 2017 were 78.8% and 83.5%, respectively. The survival rate of the 2016 normal production cohort was 63.8%. The segregation pattern of the resistant allele showed 1 (+/+): 2 (+/−): 1 (−/−) in G2, and genotype frequency of Pma4_014 was calculated using approximately 200 individuals collected from 2016 and 2017 G2 populations before and after the field trial. Numbers of each genotype were estimated based on the number of introduced and surviving fish. Individuals with the resistant allele, (+/+) and (+/−), showed over 80% of the estimated survival rates, but individuals without the resistant allele (−/−) showed lower survival (50%) in both years. This result strongly suggests that the resistant allele dominantly affects RSIVD resistance, and the newly developed G2 population is useful for further propagation of RSIVD-resistant red sea bream fingerlings.

We detected an earthy/musty off-flavor in the flesh of red sea bream Pagrus major cultured under low saline water conditions using two types of recirculating aquaculture systems (RAS). The compound causing the off-flavor was identified, and we investigated how the ultraviolet (UV) equipment of the RAS affected the concentrations of off-flavor compounds in fish flesh and rearing water. Odor intensity was lower in the muscle of fish from RAS equipped with a UV device (UV+) compared to RAS with no UV device (UV−). In total, 107 volatiles were identified in the muscles of fish from each RAS and sea cage (control) by gas chromatography–mass spectrometry. The volatile compounds previously reported to be responsible for the off-flavor were not detected. The odorous compound that caused the off-flavor in red sea bream flesh from both of the RAS was identified by gas chromatography–olfactometry as 2-methylisoborneol (2-MIB). The concentrations of 2-MIB in the fish flesh and rearing water were lower in UV+ compared to UV−. In conclusion, the problem of an off-flavor in marine fish reared in RAS-type temporary rearing tanks with diluted seawater will likely increase as more marine fish species are reared under these conditions.

Two types of chromophobic granules [type 1, ordinary type (oβG-1^{Ld})； type 2, extraordinary type (eoβG-2^{Ld})] were observed in the neutrophils of red seabream Pagrus major infected with Lamellodiscus spp. The oβG-1^{Ld} showed similar morphology and cytochemistry to type 1 ordinary chromophobic granule (oβG-1) from non-infected fish: Chromophobic, simple morphology (without stratified structure), positive reaction to peroxidase (PO) and Sudan black B (SBB), and lack of lysozomal enzymes. The eoβG-2^{Ld} was stratified granule with two-layer structure [inner eosinophilic layer (L0) and outer chromophobic layer (L1)]. Several lysozomal enzymes [acid phosphatase (AcP), β-glucuronidase, α-naphtyl acetate esterase, α-naphtyl butyrate esterase, naphthol AS-D chloroacetate exterase (CAE)] were detected in the L0, however, AcP and CAE were localized in the small central area of L0. These findings indicate that the L0 of eoβG-2^{Ld} consists of L0-0 (inner part in L0； AcP and CAE positive) and L0-1 (outer part of L0-0). The L1 showed positive reaction to SBB and PO. Spot formation, a curious phenomenon appeared in PO-stained type 2 ordinary chromophobic granule (oβG-2； positive L1 and negative L0) from non-infected fish, was not observed in eoβG-2^{Ld}.

Moina macrocopa (Straus, 1820), was cultured on different animal manures (i.e. chicken manure, Pig manure and cow manure) and food waste to detect the influence of these food sources on its mass production. Then M. macrocopa was used as feed for the rearing of Pagrus major larvae and its effects on their performance was studied. At first, the experiment was carried out for 21 days to identify the best culture medium for M. macrocopa. Food waste treatment resulted in higher density of M. macrocopa compared to other animal wastes. The percentage of unsaturated fatty acids was also significantly higher in the food waste treatment. Subsequently, we compared the effect of M. macrocopa cultured on food waste, Artemia and artificial diet on rearing the larvae of Pagrus major, red sea bream. The feeding time differed for each treatment, but the feeding number and amount were adjusted for all treatments. Results showed that larvae fed with M. macrocopa showed higher survival rates, growth rates, stress tolerance rate and DHA/EPA ratio than larvae fed with Artemia and artificial diet. The present results revealed that M. macrocopa cultured on food waste has an outstanding food potential and a probable substitute for Artemia. Keywords: Moina macrocopa, Food waste, Fatty acid content, Artificial diet, Pagrus major, Larvae

We report that the morphology and cytochemistry of neutrophils from a red seabream Pagrus major infected with numerous Ceratothoa verrucosa (Cv). First, the host was artificially infected with two Cv (adults； usual parasitism), however, finally with 34 Cv (26 Cv were located in gill cavity) except for adults (superunusual parasitism). By observation of blood smear from the host, we noticed the appearance of new type of neutrophil ‘third type； neutrophil-3^{rd}’. The neutrophil-3^{rd} had two types of granules. Both granule types had similar morphology but different cytochemical characteristics to those of ordinary chromophobic granules (oβG-1, oβG-2) of neutrophil from the fish without Cv. In present paper, we call the two granule-types of neutrophil-3^{rd} extraordinary chromophobic granules (type 1, eoβG-1^{Cv3rd} ； type 2, eoβG-2^{Cv3rd}). The eoβG-1^{Cv3rd} showed chromophobic, simple morphology (without stratified structure), peroxidase positive and lack of lysozomal enzymes. On the other hand, the eoβG-2^{Cv3rd} was stratified granule with two-layer structure [inner eosinophilic layer (L0) and outer chromophobic layer (L1)]. Lysozomal enzymes (acid phosphatase, β-glucuronidase and esterases) and peroxidase (PO) were localized in L0 and L1, respectively. However, the lysozomal enzymes were limited in the small central area of L0. Therefore, L0 was interpreted as two-layer structure: L0-0 (inner part in L0) and L0-1 (outer part in L0). Spot formation, a curious phenomenon appeared in PO-stained oβG-2 (positive L1 and negative L0), was also observed in eoβG-2^{Cv3rd}. Both types of extraordinary granules showed negative reaction to Sudan black B, oil red O and Sudan III, however, entire L0 of eoβG-2^{Cv3rd} was stained blue with hematoxylin for counter stain in these preparations.