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The limited localization and penetration of monoclonal antibodies (mAb) into solid tumors restricts their antitumor efficacy. Here, we describe a solid tumor–targeting antibody with enhanced tumor penetration activity. We designed a 22-residue peptide (A22p), which was extracted from the C-terminal basic region of semaphorin 3A (Sema3A) but modified to have higher affinity with neuropilin receptors (NRP), and genetically fused it to the C-terminus of Fc of human immunoglobulin G1 via a 15-residue (G4S)3 linker, generating Fc-A22p, for the bivalent binding to NRPs. In contrast to Fc or the monovalent A22p peptide alone, Fc-A22p homed to tumor vessels and induced vascular permeability through VE-cadherin downregulation and penetrated tumor tissues by interacting with NRPs in mice bearing human tumor xenografts. We extended the Fc-A22p platform by generating mAb-A22p antibodies of two clinically approved solid tumor–targeting mAbs, the anti-EGF receptor mAb cetuximab (erbitux), and the anti-Her2 mAb trastuzumab (herceptin). The mAb-A22p antibodies retained the intrinsic antigen binding, natural Fc-like biophysical properties, and productivity in mammalian cell cultures, comparable with those of the parent mAbs. In mouse xenograft tumor models, the mAb-A22p antibodies more efficiently homed to tumor vessels and spread into the extravascular tumor parenchyma, which significantly enhanced antitumor efficacy compared with the parent mAbs. Our results suggest that mAb-A22p is a superior format for solid tumor–targeting antibodies due to its enhanced tumor tissue penetration and greater antitumor efficacy compared with conventional mAbs. Mol Cancer Ther; 13(3); 651–61. ©2014 AACR.

PDF - 2613K, Figure S1: Structural analysis of NRP1, NRP2, and Sema3A, and their interactions to facilitate the understanding of the interactions between Sema3A and NRP1/2. Figure S2: SDS-PAGE analyses of the purified proteins showing that NRP1/2-b1b2 domains, Fc-A22, and Fc-A22p are well prepared. Figure S3: FACS and confocal fluorescence data showing that Fc-A22p, but not Fc, specifically interacts with cell-surface expressed NRP1/2 undergoing cellular internalization. Figure S4: Cell viability data showing that Fc-A22p does not cause significant cytotoxicity on HUVECs and tumor cells. Figure S5: Tumor tissue analyzing data showing that Fc-A22p homes to tumor, induces vascular permeability, and downregulates VE-cadherin in endothelial cells and E-cadherin in tumor cells. Figure S6: Confocal fluorescence data showing that Fc-A22p causes downregulation of VE-/E-cadherin and their dissociation from F-actin cytoskeleton resulting in disruption of cell-cell contacts. Figure S7: Data showing the expression and biochemical characterization of mAb-A22p antibodies, cetuximab-A22p and trastuzumab-A22p, compared with the parent mAbs. Figure S8: Cell proliferation and Western blotting data showing that cetuximab-A22p exerts in vitro biological activities comparable to those of cetuximab in A431 and FaDu tumor cells. Figure S9: Human tumor xenografted mouse data showing that mAb-A22p antibodies show increased tumor homing and penetration and thus superior in vivo anti-tumor efficacy, compared with the parent mAbs. Table S1: SPR data showing the kinetic binding parameters for the interactions of Fc-A22, Fc-A22p, VEGF165, and Sema3A with soluble NRP1-b1b2 and NRP2-b1b2 fragments. Table S2: Purification yields of Fc proteins and antibodies obtained from transient expression in HEK293F cells. Table S3: SPR data showing the kinetic binding parameters for the interactions of mAbs and mAb-A22p antibodies with the indicated proteins. Supplementary Materials and Methods: Detailed description of Reagents; Recombinant protein expression and purification; Construction, expression, and purification of antibodies; Binding analysis by ELISA; Surface plasmon resonance (SPR); Flow cytometric analysis; Cell proliferation assay; Western blotting and immunoprecipitation; Co-localization studies by confocal immunofluorescence microscopy; RNA Interference; Ex vivo tumor penetration assays.

Reproductive cycle of the blue-striped angelfish, Chaetodontoplus septentrionalis were histologically investigated. Fish were monthly collected in the coastal waters of Munseom, Seogwipo, Jeju-Island from February to December 2018. The gonadosomatic index (GSI) increased from May and maintained high values in August. The reproductive cycle of female fish can be classified by the characteristics observed during gonadal development as followed: growing stage (November to June), early mature stage (May to June), mature and spawning stage (June to September), and degenerative and recovery stage (September to December). In the male, testicular development period was similar to that of ovarian development period, but mature and spawning period was one month longer from June to October. Fecundity of mature female ranged from 4,601 to 22,840 and was correlated positively with total length and body weight. The histological analysis of gonadal development indicated that the C. septentrionalis was summer-spawning type and is considered a multiple spawner during spawning season.

; Lee

Rosemary essential oil was evaluated for antifungal potentiality against six major ginseng pathogens: Sclerotinia sclerotiorum, Sclerotinia nivalis, Cylindrocarpon destructans, Alternaria panax, Botrytis cinerea, and Fusarium oxysporum. The in vitro fungicidal effects of two commonly used fungicides, namely mancozeb and fenhexamid, and the volatile organic compounds (VOCs) of Trichoderma koningiopsis T-403 on the mycelial growth were investigated. The results showed that rosemary essential oil is active against all of the pathogenic strains of ginseng root rot, whereas rosemary oil displayed high ability to inhibit the Sclerotinia spp. growth. The highest sensitivity was S. nivalis, with complete inhibition of growth at 0.1% v/v of rosemary oil, followed by Alternaria panax, which exhibited 100% inhibition at 0.3% v/v of the oil. Minimum inhibitory concentrations (MICs) of rosemary oil ranged from 0.1 % to 0.5 % (v/v). Chemical analysis using GC-MS showed the presence of thirty-two constituents within rosemary oil from R. officinals L. Camphore type is the most frequent sesquiterpene in rosemary oil composition. Mancozeb and fenhexamid showed their highest inhibition effect (45% and 30%, respectively) against A. panax. T. koningiopsis T-403 showed its highest inhibition effect (84%) against C. destructans isolate. This study may expedite the application of antifungal natural substances from rosemary and Trichoderma in the prevention and control of phytopathogenic strains in ginseng root infections.

As a preliminary investigation into the effects of environmental factors on gonadal development using adaptive physiological characteristics, we examined the involvement of photoperiod and water temperature in the ovarian development of the redspotted grouper, Epinephelus akaara. During October 2014 (non-breeding season), the photoperiod and water temperature were adjusted to 12L:12D and 18°C respectively. Every 3 weeks, daylight was increased as follows a 13L:11D and 14L:10D in the photo-thermal treatment group, and the control group was maintained under natural conditions. After 9 weeks, water temperature was increased to 23°C in the photo-thermal treatment group. In photo-thermal treatment group, GSI increased significantly after 9 weeks (1.36 ± 0.19) and 12 weeks (3.31 ± 0.20) compared with the control group. At 12 weeks after photo-thermal treatment, all fishes matured into ovaries containing late vitellogenic oocytes. The expression of FSH β and LHβ mRNAs also increased significantly. However, in the control group, the ovarian development was maintained at an immature stage. Based on these results, we demonstrate that exposure to long photoperiod and high water temperature can induce the sexual maturation of immature red-spotted grouper during the non-breeding season.

The aim of this study was to investigate the expression patterns of ocular melatonin in the annual reproductive cycle of the female grass puffer. Spawning season of the female grass puffer is from June to July in Jeju, South Korea. Time-resolved fluoroimmunoassay revealed that levels of ocular melatonin, which show an annual change, peaked in May (spawning season). Additionally, expression of reproductive-related genes also showed annual patterns: GnRH1 peaked in August, GnRH2 peaked in February, GnRH3, Kiss2, and LPXRFa peaked in November. These results suggest that ocular melatonin may be related to the annual reproductive cycle in the grass puffer. To better understand the photic regulation of AANAT1a mRNA in the retina, we observed the nocturnal pattern of ocular melatonin levels daily, which shows a nocturnal pattern in both short photoperiod (SD) and long photoperiod (LD) conditions. In the brain, AANAT2 mRNA also shows a nocturnal pattern in both SD and LD; however, the time of peak expression of AANAT2 mRNA was unchanged in both conditions. Following intraperitoneal injection of melatonin for 2 weeks, expression of GnRH2 and LPXRFa mRNA in the brain significantly increased, while that of Kiss2 mRNA was decreased, suggesting that melatonin has a reproduction-related effect. Furthermore, under SD and LD conditions for 14 weeks, the gonadosomatic index more increased and the maturity of the ovary progressed under LD compared with those under SD, suggesting that the SD photoperiodic signal inactivated ovarian development. These results indicate that the ocular melatonin may have a possible role in the reproductive endocrinology of the grass puffer.

To produce healthy and stable seed production, we need to obtain information and understand vision that affects behavior of red spotted grouper. We examined their expression and retinal development during the juvenile development. Short-wavelength sensitive opsin (SWS2), a cone photoreceptor, began to be expressed from lens and ear vesicle formation stage and its expression increased until 10 days after hatching (dah). In case of middle-wavelength sensitive opsin (MWS), its expression was detected at 3 dah and reached the highest level at 21 dah. The expression of long-wavelength sensitive opsin (LWS) was first observed from 3 dah and their expression decreased thereafter. Rhodopsin, a rod photoreceptor, was found to be expressed from 2 dah and its expression reached the highest level at 50 dah. The outer nuclear layer (ONL), inner nuclear layer (INL) and ganglion cell layer began to differentiate at 2 dah, while choroid first appeared at 4 dah so that the eyes became black. These results indicate that the development of retina mostly completes around 4 dah. It seems that the development of the retina and the expression of the opsin genes are closely related to the behavior such as hunting prey, considering that the timing of the completion of the development of the retina, the timing of gene expression, and the timing of completion of yolk absorption are similar.

This study examined the effects of different photoperiod conditions on olive flounder (Paralichthys olivaceus), a commercially important species in Korea. Daily variations in the expression of mRNA for the growth-related genes ary-lalkylamine N-acetyltransferase2 (AANAT2), preprosomatostatin1 (PSS1), and growth hormone (GH) were examined under a 12 h light:12 h dark photoperiod. All the genes were expressed at higher level during the dark period. Melatonin injections increased the expression of GH, but did not significantly affect the expression of PSS. Under short-day conditions (10 h:14 h), the fish gained more weight than under long-day conditions (14 h:10 h). A long nighttime induced melatonin secretion and increased the expression of GH mRNA, promoting weight gain in this species. Therefore, we thought that the long day condition in raising olive flounder may be effective in inducing body growth.

Fish shows great difference in growth rate between individuals during larval development and early growth. This difference seriously reduces the production efficiency in fish culture. Growth hormone (GH)/Insulin-like growth factor 1 (IGF1) system is said to play some pivotal roles in fish growth. In this study, we investigated differences of GH, IGF1 and GHR gene expressions in juvenile red spotted grouper (Epinephelus akaara) with different growth performance. Red spotted groupers were reared under the same environmental condition (water temperature 24±1℃, natural light) for 96 days after hatching. They were divided into 3 groups by size (fast growing, middle growing and slow growing groups: FGG, MGG, and SGG, respectively). RNA was extracted from the brain, liver and muscle tissues from each group, and target gene expression was examined by real-time PCR. In the brain with pituitary gland, expression of GH gene in FGG was significantly higher than the expression in SGG, but the expression of IGF1 and GHR genes in the muscle was highest in SGG. Difference of GHR and IGF1 mRNA in the liver between groups with different growth performance was less clear than that in other tissues, although level of IGF1 mRNA was higher in SGG than in MGG. These results suggest that hormonal governing of growth is not the same in fast growing and slow growing fish, and size grading could cause a shift of hormonal state and growth pattern in this species.

We determined complete nucleotide sequences of the mitochondrial genome of two individuals of the Red-spotted grouper, Epinephelus akaara (Perciformes, Serranidae), caught in South Korea. The mitochondrial genome had 16,795 base pairs (bp) and 13 protein-coding genes, 2 ribosomal RNAs, 22 transfer RNAs, and a noncoding control region. The two mt genomes were highly homologous (99.71% similarity). The two mt genomes of E. akaara analyzed in this study were found in Clade I with those of E. awoara, E. fasciatomaculosus, E. sexfasciatus, E. diacanthus, E. sticus, and E. morio. Here, we reported the complete mt genome sequence of E. akaara, suggesting that this may use in phylogenetic studies of Epinephelus.

Background Fish body color is one of the major factors that determine the commercial value of farmed fish, to understand for coloration mechanisms. The expression of melanin-related genes is according to the developmental stage and light intensity in the red spotted grouper, Epinephelus akaara. Methods To investigate changes in melanin formation and melanin-related genes in the larval development stage, fish larvae were reared from fertilized egg stage to 50 days after hatching (DAH). Experiment of body color changes was performed under each different light intensity conditions. Melanin-related genes expression was analyzed by real-time qPCR, and body coloration difference was represented by RGB (red, green, blue) code value. Results Expression levels of melanin-concentrating hormone (MCH) mRNA, pro-opiomelanocortin (POMC) mRNA, and melanocortin 1 receptor (MC1R) mRNA were at their highest 5 DAH (days after hatching). Expression levels of agouti-signaling protein (ASIP) mRNA were at their highest 10 DAH. Results of body color changes according to changes in light intensity conditions showed that the expression level of MCH mRNA in the 2000 lx group was the highest at 6 weeks. The expression levels of POMC mRNA and MC1R mRNA in the 1000 lx group were at their lowest at 9 weeks. RGB color code value under different light intensities were the brightest under 1000 lx and the darkest under 0 lx. Conclusion Our research suggests that POMC mRNA and MC1R mRNA in the red spotted grouper are involved in melanin synthesis, and these genes are thought to be controlled by light intensity. To better understand the molecular mechanism of coloration in the red spotted grouper, further studies are needed to clarify the relationship between melanophore development and melanin-related genes.

Rosemary essential oil was evaluated for antifungal potentiality against six major ginseng pathogens

Most wrasses are protogynous species that swim to feed, reproduce during the daytime, and bury themselves under the sandy bottom at night. In temperate and subtropical wrasses, low temperature influences emergence from the sandy bottom in the morning, and induces a hibernation-like state in winter. We cloned and characterized the prohormone complementary DNAs (cDNAs) of arginine vasotocin (AVT) and isotocin (IT) in a temperate wrasse (Halichoeres tenuispinis) and examined the effects of day/night and temperature on their expression in the diencephalon, because these neurohypophysial peptides are related to the sex behavior of wrasses. The full-length cDNAs of pro-AVT and pro-IT were 938 base pairs (154 amino acids) and 759 base pairs (156 amino acids) in length, respectively. Both pro-peptides contained a signal sequence followed by the respective hormones and neurophysin connected by a Gly-Lys-Arg bridge. Reverse-transcription polymerase chain reaction (RT-PCR) revealed that pro-AVT mRNA expression was specifically observed in the diencephalon, whereas pro-IT mRNA expression was seen in the whole brain. Quantitative RT-PCR revealed that the mRNA abundance of pro-AVT and pro-IT was higher at midday (zeitgeber time 6; ZT6) than at midnight (ZT18) under 12 h light and 12 h darkness (LD 12:12) conditions, but not under constant light. Intraperitoneal injection of melatonin decreased the mRNA abundance of pro-AVT, but not of pro-IT. When fish were reared under LD 12:12 conditions at 25, 20, and 15 degrees C, day high and night low mRNA expressions of pro-AVT and pro-IT were maintained. A field survey revealed seasonal variation in the number of swimming fish at observatory sites; many fish emerged from the sandy bottom in summer, but not in winter, suggesting a hibernation-like state under the sandy bottom under low temperature conditions. We conclude that the day-night fluctuation of pro-AVT and pro-IT mRNA abundance in the brain is not affected by temperature and repeated under the sandy bottom in winter.

Skeletal deformities are significant problems that affect the growth and commercial value of fish reared in hatcheries. However, studies of bone metabolic process related to skeletal deformities are limited. We investigated the potential correlation between bone deformities and plasma calcium, phosphorus, and estradiol-17β levels in reared red spotted grouper (Epinephelus akaara) juveniles. We collected E. akaara frys from private farms at 110, 140, 180 and 300 days after hatching (DAH), and classified the normal and deformed fish by observing their external shape and inner frame by soft X-ray. We also analyzed the calcium, phosphorous, and estradiol-17β levels in their plasma. A comparison between normal and deformed fish, indicated that calcium and estradiol-17β levels were higher in deformed fish than in the normal at 180 and 300 DAH. The level of phosphorus was also higher in deformed individuals than in normal fish, but only at 300 DAH. These results suggest that skeletal deformities are associated with increases in plasma calcium, phosphorus, and estradiol-17β levels.

Survival of embryos largely depends on yolk processing during early development. Proteolytic enzymes, cathepsin B & D (ctsb & ctsd) are known to have some important roles in yolk processing of various fish species. Mature female red spotted groupers were injected with human chorionic gonadotropin (HCG) to induce ovulation. The fertilized eggs and embryos were sampled at 0, 4 and 24 HPF (hours post fertilization). Survivals of each groups of embryos were checked at 24 and 48 HPH (hours post hatching). Transcripts of ctsb & ctsd showed the highest level at 0 HPF and relatively high at 4 HPF, but greatly decreased at 24 HPF. In bad egg quality group (BE, embryos survived until 24 HPH), transcript level of ctsb at 4 HPF were significantly lower than the transcript level at the same stage in good egg quality group (GE, embryos survived until 48 HPH) while no significant change of ctsb transcript level was observed at 0 or 24 HPF between BE and GE. Transcript level of ctsd was decreased at 24 HPF, but the difference was not as strong as the case of ctsb transcript. These results suggest that maternal ctsb transcript rather than ctsd transcript is likely to be involved in egg quality resulting in the difference of survival rate of embryos at early developmental period in this species.

Somatostatin (SS) and growth hormone-releasing hormone (GHRH) are primary factors regulating growth hormone (GH) secretion in the pituitary. To date, it remains unknown how this rhythm is controlled endogenously, although there must be coordination of circadian manners. Melatonin was the main regulator in biological rhythms, and its secretion has fluctuation by photic information. But relationship between melatonin and growth-related genes (ghrh and ss) is unclear. We investigated circadian rhythms of melatonin secretion, ghrh and ss expressions, and correlation between melatonin with growth-related genes in tiger puffer Takifugu rubripes. The melatonin secretion showed nocturnal rhythms under light and dark (LD) conditions. In constant light (LL) condition, melatonin secretion has similar patterns with LD conditions. ss1 mRNA was high during scotophase under LD conditions. But ss1 rhythms disappeared in LL conditions. Ghrh appeared opposite expression compared with melatonin levels or ss1 expression under LD and LL. In the results of the melatonin injection, ghrh and ss1 showed no significant expression compared with control groups. These results suggested that melatonin and growth-related genes have daily or circadian rhythms in the tiger puffer. Further, we need to know mechanisms of each ss and ghrh gene regulation.

This study investigated the effect of different photoperiods (14L: 10D, 12L:12D and 10L:14D) on the gonadal development and GtH mRNA expression in the pituitary of damselfish. The results showed that gonadosomatic index (GSI) was significantly lower in shot photoperiod (10L:14D), in comparison with other photoperiodic group during the spawning season. After 60 days treatment, histological analysis of gonad tissue showed that the gonad of 10L:14D and 12L: 12D treatment groups were resting phase with spermatogonia and perinucleolus stage oocytes but the gonad of 14L:10D treatment group was still ripe phase with spermatozoa and mature stage oocyte. The FSHβ and LHβ mRNA expression in pituitary drastically decreased shot photoperiod treatment from July (spawning period). These results suggest that photoperiod is considered to be the most effective environmental factor in controlling the reproductive cycle of damselfish.

BACKGROUND Studies effectively examining temporal patterns of papillary thyroid cancer (PTC) recurrence are currently lacking. The purpose of this study was to examine sites of PTC recurrence, interval from initial treatment to recurrence, and changing patterns of recurrence during long-term follow-up. METHODS Records of 134 patients with PTC recurrence were analyzed retrospectively. RESULTS The most common site of initial recurrence was the lateral neck. In 6.7% of patients, distant metastases occurred as initial recurrences. In 74%, recurrences occurred within the first 5 years of surgery, whereas in 5.2%, and 1.5%, recurrences occurred between 10 to 20 years and after 20 years of surgery, respectively. Mean time to recurrence and distant metastasis was 48.2 months and 92.5 months, respectively. Male sex (p = .002), size (p < .001), N classification (p < .001), and recurrence frequency (p = .049) were prognostic factors for distant recurrence. CONCLUSION Men with PTC tumors larger than 2 cm, lateral neck node metastasis, and multiple local recurrences should be scrutinized for distant metastasis even after 10 years. © 2017 Wiley Periodicals, Inc. Head Neck 39: 767-771, 2017.

This study examined the effects of different photoperiod conditions on olive flounder (Paralichthys olivaceus), a commercially important species in Korea. Daily variations in the expression of mRNA for the growth-related genes ary­lalkylamine N-acetyltransferase2 (AANAT2), preprosomatostatin1 (PSS1), and growth hormone (GH) were examined under a 12 h light:12 h dark photoperiod. All the genes were expressed at higher level during the dark period. Melatonin injections increased the expression of GH, but did not significantly affect the expression of PSS. Under short-day conditions (10 h:14 h), the fish gained more weight than under long-day conditions (14 h:10 h). A long nighttime induced melatonin secretion and increased the expression of GH mRNA, promoting weight gain in this species. Therefore, we thought that the long day condition in raising olive flounder may be effective in inducing body growth.

In this study, we investigated the characteristics of CCK-producing cells and mucus-secreting goblet cells with respect to stomach fish and stomachless fish of the Gobiidae in order to provide a basis for understanding the digestive physiology. Hairychin goby (Sagamia geneionema), which is stomachless fish, the numbers of mucus-secreting goblet cells is highest in the posterior intestine portion (P

This study monitored the morphological development of embryo, larvae and juvenile yellowtail kingfish, Seriola lalandi, for their aquaculture. The fertilized eggs obtained by natural spawning were spherical shape and buoyant. Fertilized eggs were transparent and had one oil globule in the yolk, with an egg diameter of 1.35 ± 0.04 mm and an oil globule diameter of 0.32 ± 0.02 mm. The fertilized eggs hatched 67–75 h after fertilization in water at 20 ± 0.5°C. The total length (TL) of the hatched larvae was 3.62 ± 0.16 mm. During hatching, the larvae, with their mouth and anus not yet opened. The yolk was completely absorbed 3 days after hatching (DAH), while the TL of post-larvae was 4.72 ± 0.07 mm. At 40 DAH, the juveniles had grown to 30.44 ± 4.07 mm in TL, body depth increased, the body color changed to a black, yellow, and light gray-blue color, and 3–4 vertical stripes appeared. At 45 DAH, the juveniles were 38.67 ± 5.65 mm in TL and 10.10 ± 0.94 mm in body depth. The fish were green with a light orange color, with 7 faint green-brown stripes on the sides of their body. At 87 DAH, the juveniles had grown to 236.11 mm in TL, 217.68 mm in fork length, and 136.5 g in weight. The fish resembled their adult form, with a light yellow-green body color, loss of the pattern on the sides of their body, and a yellow coloration at the tip of the caudal fin.

Streptococcus iniae is a causative agent of hemorrhagic septicemia in olive flounder, Paralichthys olivaceus, in Korea, resulting in serious economic losses. As a preventive measure, M VAC INIAE (Mastuken, Japan) was prepared from the S. iniae F2K strain and tested against the SI-36 strain prevalent on flounder fish farms on Jeju Island, Korea. F2K had a serotype of 38 (–) and SI-36 38 (+). The vaccine recognized both serotypes. It showed a very high effective immune response against S. iniae; the challenge test using the S. iniae SI-36 strain resulted in a relative percent survival (RPS) of 85.7-87.0% 2 weeks after vaccination and 71.0-80.0% 6 months after vaccination. Field vaccination and clinical challenge tests were performed at local Jeju aquafarms with S. iniae SI-36. These showed significantly reduced cumulative mortality when compared to the control group with RPS rates that ranged between 71-80%. Hence, the present study suggests that this vaccine showed a significant immune response against S. iniae and could be applied in commercial aquafarms as a therapeutic agent against â-hemolytic streptococcosis in cultured P. olivaceus. CORRESPONDING AUTHOR: Y.U. Jeong, D. Subramanian, D.H. Kim, S.H. Park, M.S. Heo [+] Marine Pathogenic Microbes Lab, Department of Aquatic Biomedical Sciences, School of Marine Biomedical Sciences, Jeju National University, Jeju 690-756, South Korea e-mail: msheo@jejunu.ac.kr Y.-H. Jang Jeju Special Self-Governing Province Ocean and Fisheries Research Institute, Pyoseon-myeon, Segwipo-si, Jeju 697-914, South Korea K. Park Department of Aquatic Life Medicine, Kunsan National University, Kunsan 573-701, South Korea Y.D. Lee Marine and Environmental Institute, Jeju National University, Jeju 690-814, South Korea Y.U. Jeong and D. Subramanian contributed equally to this work.

This study describes results on sexual maturation and characteristics of natural spawned eggs to develop a method for the production of stable, healthy fertilized eggs from captive-reared yellowtail kingfish, Seriola lalandi. A total of 59 yellowtail kingfish were captured off the coast of Jeju Island, after which the broodstock was cultured in indoor culture tank (100 m(3)) until they were 6.1-14.9 kg in body weight. As part of the rearing management for induced sex maturation, the intensity of illumination was maintained at 130 lux. The photoperiod (light/dark; L/D) was set to a 12 L/12 D from October 2013 to January 2014, and 15 L/9 D from February 2014 to June 2014. Feeds comprised mainly EP (Extruded Pellets), with squid cuttlefish added for improvement of egg quality, and was given from April to June 2014. The first spawning of yellowtail kingfish occurred in May 3, 2014, at a water temperature of 17.0°C. Spawning continued until June 12, 2014, with the water temperature set at 20.5°C. Time of spawning was 26 times at this period. The total number of eggs that spawned during the spawning period was 4,449×10(3). The buoyant rate of spawning eggs and fertilization rate of buoyant eggs during the spawned period were 76.1% and 100%, respectively. The diameters of the egg and oil globule were 1.388 ± 0.041 mm and 0.378 ± 0.029 mm, respectively, which was higher in early eggs than in those from late during the spawned period.

It takes quite a long time for the grouper to spawn. In the case of red spotted grouper (Epinephelus akaara), at least three to four years of rearing is usually required to reproduce them for the first time. Reproductive control techniques can be applied to repress, delay or advance the onset of puberty. Thus, they can be used to accelerate the process of selective breeding in this species. The present study investigated whether alterations of rearing water temperature (WT) can advance the onset of puberty in the red spotted grouper. Juvenile red spotted grouper (110 DAH, 7.25 ± 0.5 cm, 6.45 ± 1.5 g) were randomly divided into 4 groups and reared for approximately 10 months (from Nov. 2014 to Aug. 2015) at four different WT: natural condition (12.6-19.5°C), 20 ± 0.5°C, 24 ± 0.5°C and 28 ± 0.5°C treatment. When they were reared at 24 ± 0.5°C or 28 ± 0.5°C WT, sexually mature individuals appeared within 12 months after hatching during their breeding season (Jul. to Aug.). The mRNA levels of reproduction-related genes such as Kisspeptin, GnRH, FSHβ and LHβ were higher in the 24 ± 0.5°C and 28 ± 0.5°C treatment group than the other groups (P< 0.05). Mature yolk stage oocytes (≥300 μm diameter) were found in the ovaries of female red spotted grouper reared at 24 ± 0.5°C or 28 ± 0.5°C groups, while only oogonia were found at natural condition and peri-nucleolus stage oocytes were observed at 20 ± 0.5°C group, respectively. The one-year-old mature females ovulated 6-10 ml of eggs that corresponded to 10% of their body weight. In artificial fertilization performed at 24 ± 0.5°C WT, the fertilization and hatching rates were determined to be 95% and 97%, respectively. This is the first report demonstrating that rearing at 24 or 28℃ WT can significantly advance the onset of puberty in the red spotted grouper.