Changes in Behavior and Diel Melatonin Secretion Toward Estivation in Western Sand Lance, Ammodytes japonicus (Uranoscopiformes, Ammodytidae).

Western sand lance, Ammodytes japonicus, is known to have an estivation period, in which they cease feeding and stay in the sand from early summer to late autumn, followed by gonadal maturation. During the feeding period prior to estivation, they swim in daytime and spend the night in the sand. Before they start swimming, they show a typical behavior of head-exposing from the sand, which is likely to be related to foraging and predation avoidance. Our previous study revealed that melatonin regulates such diel behavior of this species. To elucidate the mechanisms of behavioral regulation throughout the life cycle of this sand lance, the present study examined the changes in behavior and melatonin secretion toward the estivation period. Both head-exposing and swimming behaviors were frequently observed at the transition period toward estivation. On the other hand, occurrence of these behaviors was suppressed just before entering estivation. Subsequently, it was found that plasma melatonin concentration was about three times higher at night than in daytime in the non-estivation period, while it was retained at high levels throughout the day in the estivation period. These results indicate that diurnal swimming behavior of sand lance from the feeding to estivation periods is associated with the daily cycle of melatonin secretion.

Characterization of eight types of 17ß-hydroxysteroid dehydrogenases from the Japanese sardine Sardinops melanostictus: The probable role of type 12a in ovarian estradiol synthesis.

17ß-hydroxysteroid dehydrogenases (Hsd17bs) play a critical role in sex steroid biosynthesis. Although multiple types of Hsd17b have been found in fish, there is limited research on their expression and function. Recently, we succeeded in identifying eight types of Hsd17b (types 3, 4, 7, 8, 10, 12a, 12b, and 14) by RNA sequencing in the Japanese sardine Sardinops melanostictus, a commercially important clupeoid fish; however, a homologous sequence of Hsd17b1, which catalyzes the key reaction of estradiol-17ß (E2) synthesis, was absent. Here, we aimed to identify the Hsd17b type that plays a major role in E2 synthesis during ovarian development in Japanese sardine. The cDNAs encoding those eight types of Hsd17b were cloned and sequenced. The expressions of hsd17b3, hsd17b12a, and hsd17b12b were higher in ovary than in testis. In particular, hsd17b12a was predominantly expressed in the ovary. Expression of hsd17b3, hsd17b4, hsd17b12a, and hsd17b12b in the ovary increased during ovarian development. The enzymatic activities of Hsd17b3, Hsd17b12a, and Hsd17b12b were evaluated by expressing their recombinants in human embryonic kidney 293T cells. Hsd17b12a and Hsd17b12b catalyzed the conversion of androstenedione (AD) to testosterone (T) and estrone (E1) to E2. The results of in vitro bioassays using sardine ovaries indicated that E2 is synthesized from pregnenolone via AD and T, but not E1. These results suggest that Hsd17b12a plays a major role in E2 synthesis in sardine ovary by catalyzing the conversion of AD to T.

Development of sandwich enzyme-linked immunosorbent assays for chub mackerel Scomber japonicus gonadotropins and regulation of their secretion in female reproduction.

The pituitary gonadotropins (Gths), follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh), play critical roles in regulating gonadal development and sexual maturation in vertebrates. We developed non-competitive enzyme-linked immunosorbent assays (ELISAs) to measure Fsh and Lh in chub mackerel Scomber japonicus, which is a commercially important scombrid species. Mouse monoclonal antibodies specific for Fsh and Lh, and a rabbit polyclonal antibody against both Gths were produced by immunization with hormones purified from chub mackerel pituitaries. These monoclonal and polyclonal antibodies were used as capture and detection antibodies in the developed sandwich ELISAs. The ELISAs were reproducible, sensitive, and specific for chub mackerel Fsh and Lh. Parallelism between the standard curve and serial dilutions of chub mackerel serum and pituitary extract was observed for both Fsh and Lh ELISAs. Comparison between vitellogenic and immature females revealed that Fsh is secreted during vitellogenesis and Lh is barely released during immaturity. After gonadotropin-releasing hormone analog (GnRHa) injection, vitellogenic females showed increases in serum Lh, whereas serum levels of Fsh did not vary. Moreover, the serum steroid profiles revealed that estradiol-17ß was continuously produced after GnRHa treatment, whereas 17,20ß-dihydroxy-4-pregnen-3-one secretion was transiently induced. These results indicate that, in vitellogenic females, GnRHa stimulates the release of Lh, but not Fsh, which results in acceleration of vitellogenesis and induction of oocyte maturation via steroid production.

Positive effects of fast growth on locomotor performance in pelagic fish juveniles.

Many laboratory experiments on aquatic vertebrates that inhabit closed water or coastal areas have highlighted negative effects of fast growth on swimming performance. Nonetheless, field studies on pelagic fishes have provided evidence of survival advantages of faster-growing individuals. To reconcile this contradiction, we examined the relationship between growth rate and swimming performance as a continuous function for juveniles of chub mackerel (Scomber japonicus) using 3D tracking analysis. For experiments, 20, 24, 27, and 30 days post-hatch individuals within the size range of 14.5-25.3 mm were used. We found that the growth-swimming (burst speed) relationship in chub mackerel was substantially positive and it was supported by morphological traits such as muscle area, which were also positively related with growth rate. This finding is consistent with field observations showing selective survival of fast-growing individuals of this species, reconciling the current contradiction between laboratory experiments and field observations. A dome-shaped quadratic curve described the relationship between growth rate and burst speed better than a linear or cubic function, suggesting that growth may trade-off with swimming performance, as reported in many previous studies, when it is extremely fast. These results, obtained from the rarely tested offshore species, strongly suggests the importance of experimental verification using animals that inhabit various types of habitats in understanding the principles underlying the evolution of growth-locomotor relationship.

Microplastic uptake and gut retention time in Japanese anchovy (Engraulis japonicus) under laboratory conditions.

To explore the impact of microplastic (MP) pollution on planktivorous fish, we examined the uptake and retention of MPs by Japanese anchovy (Engraulis japonicus) under laboratory conditions. MP uptake was size selective in adult anchovy-0.3-mm MPs were taken up in significantly larger amounts than 0.85-mm MPs-but not in juveniles. There were no significant differences in the uptake of MPs of three different colors, suggesting that anchovy do not select for MP coloration. More than 90% of the MPs were excreted within 20 h of ingestion, indicating that MP retention time is similar to the processing time of food items. Our findings suggest that Japanese anchovy tend to take up MPs that are equivalent in size to prey items, but that the impacts of MP ingestion are likely to be limited under the current state of oceanic MP contamination.

Transcriptome characterization of BPG axis and expression profiles of ovarian steroidogenesis-related genes in the Japanese sardine.

BACKGROUND: The clupeoid fishes are ecologically and commercially important fish species worldwide that exhibit a high level of population fluctuation, accompanied by alteration of reproductive traits. However, knowledge about their reproductive physiology in order to understand mechanisms underlying such population dynamics is limited. The endocrine system along with the brain-pituitary-gonadal (BPG) axis is critical for regulating reproduction. The aims of this study were to provide transcript data and genes related to the BPG axis, and to characterize the expression profiles of ovarian steroidogenesis-related genes in the Japanese sardine (Sardinops melanostictus, Clupeidae). RESULTS: RNA sequencing was performed using the sardine brain, pituitary, and gonad in both sexes. A total of 290,119 contigs were obtained and 115,173 non-redundant ORFs were annotated. The genes differentially expressed between ovary and testis were strongly associated with GO terms related to gamete production. The tissue-specific profile of the abundance of transcripts was characterized for the major regulators in the BPG axis, such as gonadotropin-releasing hormone, gonadotropin, and steroidogenic enzyme. By comparing between ovary and testis, out of eight different 17ß-hydroxysteroid dehydrogenase (Hsd17b) genes identified, higher hsd17b7 expression was found in testis, whereas higher expression of hsd17b8, hsd17b10, hsd17b12a, and hsd17b12b was found in ovary. The cDNAs encoding key endocrine factors in the ovarian steroidogenic pathway were cloned, sequenced, and quantitatively assayed. In the pituitary, follicle-stimulating hormone beta peaked during vitellogenesis, while luteinizing hormone beta peaked at the completion of vitellogenesis. In the ovary, follicle-stimulating hormone receptor and luteinizing hormone receptor were upregulated from mid- to late phase of vitellogenesis. Furthermore, three steroidogenic enzyme genes (cyp11a1, cyp17a1, and cyp19a1a) gradually increased their expression during ovarian development, accompanying a rise in serum estradiol-17ß, while 3ß-hydroxysteroid dehydrogenase and steroidogenic acute regulatory protein did not change significantly. CONCLUSIONS: This is the first report of deep RNA sequencing analysis of Japanese sardine, in which many key genes involved in the BPG axis were identified. Expression profiles of ovarian steroidogenesis-related genes provide a molecular basis of the physiological processes underlying ovarian development in the sardine. Our study will be a valuable resource for clarifying the molecular biology of clupeoid fishes.

Comprehensive Experimental System for a Promising Model Organism Candidate for Marine Teleosts.

A comprehensive experimental system for Japanese anchovy, a promising candidate model organism for marine teleosts, was established. Through the design of a rearing/spawning facility that controls the photoperiod and water temperature, one-cell eggs were continuously obtained shortly after spawning throughout the rearing period. The stages of eggs are indispensable for microinjection experiments, and we developed an efficient and robust microinjection system for the Japanese anchovy. Embryos injected with GFP mRNA showed strong whole-body GFP fluorescence and the survival rates of injected- and non-injected embryos were not significantly different, 87.5% (28 in 32 embryos) and 90.0% (45 in 50 embryos), respectively. We verified that the Tol2 transposon system, which mediates gene transfer in vertebrates, worked efficiently in the Japanese anchovy using the transient transgenesis protocol, with GFP or DsRed as the reporter gene. Finally, we confirmed that genome-editing technologies, namely Transcription Activator-Like Effector Nucleases (TALEN) and Clustered Regulatory Interspaced Short Palindromic Repeats (CRISPR)/Cas9, were applicable to the Japanese anchovy. In practice, specific gene-disrupted fishes were generated in the F1 generation. These results demonstrated the establishment of a basic, yet comprehensive, experimental system, which could be employed to undertake experiments using the Japanese anchovy as a model organism for marine teleost fish.

Food availability before aestivation governs growth and winter reproductive potential in the capital breeding fish, Ammodytes japonicus.

Capital breeders develop gametes by using energy that was stored before the spawning season. Energy is allocated to growth and reproduction, and limited food availability affects the balance of energy allocation, especially in fish that mature within a year, such as western sand lance (Ammodytes japonicus). This species aestivates without feeding until winter spawning and utilize energy stores that were accumulated prior to aestivation for maturation and spawning. This study aimed to evaluate the growth, energy storage, maturation rate, and reproduction of A. japonicus in response to food availability before aestivation. We conducted laboratory experiments in which young-of-the-year A. japonicus were fed at rates of 4% and 1% of their body weight per day; assigned as high and low ration groups, respectively. In June, body length was found to be significantly larger in the high ration group than in the low ration group, but the somatic condition did not differ significantly between the groups. Maturation rates and average fecundities were 1.0 and 6297 in the high ration group and 0.8 and 2251 in the low ration group, respectively. These results indicate that food availability before aestivation strongly governs the reproductive potential of A. japonicus, and suggest the involvement of mechanisms in the inter-annual recruitment variation in sand lance species.

Identification and distribution of three gonadotropin-releasing hormone (GnRH) isoforms in the brain of a clupeiform fish, Engraulis japonicus.

To gain a better understanding of the reproductive endocrinology of a primitive order clupeiform fish (Japanese anchovy, Engraulis japonicus), cDNAs encoding three gonadotropin-releasing hormone (GnRH) isoforms were isolated from the brain, and their distribution was analyzed using insitu hybridization (ISH). The three GnRH isoforms include GnRH1 (herring GnRH), GnRH2 (chicken GnRH-ll) and GnRH3 (salmon GnRH), and their full-length cDNAs encode 88, 86, and 89 deduced amino acids (aa), respectively. Alignment analysis of Japanese anchovy GnRH isoforms showed lower identities with other teleost fish. The major population of GnRH1 neurons was localized in the ventral telencephalon (VT) and nucleus preopticus (NPO) of the preoptic area (POA) with minor population in the anterior olfactory bulb (OB). GnRH2 neurons were restricted to the midbrain tegmentum (MT), specific to the nucleus of the medial longitudinal fasciculus (nMLF). GnRH3 neurons were localized in the olfactory nerve (ON), ventral OB, and transitional area between OB and ON. Interestingly, GnRH1 neurons were also localized in the olfactory bulb, in addition to its major population in the preoptic area. These results indicate the differential distribution of three GnRH isoforms expressed in the brain of the Japanese anchovy.

Population subdivision of Japanese flounder Paralichthys olivaceus in the Pacific Coast of Tohoku Japan detected by means of mitochondrial phylogenetic information.

This study deals with mitochondrial phylogenetic information of Japanese flounder in the Pacific coast of Tohoku Japan to estimate the genetic population subdivision that was undetectable by conventional population statistics. We determined complete sequences of mitochondrial NADH dehydrogenase subunit-2 (ND2) and subunit-5 (ND5) genes for 151 individuals from northern (Aomori and Iwate prefectures, 40-41°N) and southern (Miyagi and Fukushima prefectures, 37-38°N) waters. Samples from both waters showed high genetic diversity, including 126 haplotypes. These haplotypes were located at mixed and nested positions on an inferred phylogenetic tree, and traditional F-statistics indicated no significant population divergence (φ(ST) = -0.00335, p > 0.05), corroborating our previous study. Three variable sites, however, showed significant base composition heterogeneity between samples from the northern and southern waters (Fisher's exact-test, p < 0.01). Nucleotide substitutions at the three sites converged on an apical clade, which consisted of the five southern individuals, whereas its sister clade consisted only of the three northern individuals. This phylogenetic information corroborates previous ecological studies indicating the presence of separate stocks in the northern and southern waters.

Increased expression of kisspeptin and GnRH forms in the brain of scombroid fish during final ovarian maturation and ovulation.

BACKGROUND: Kisspeptins (Kiss) are prime players in the control of reproductive function through their regulation of gonadotropin-releasing hormone (GnRH) expression in the brain. The experimental scombroid fish, chub mackerel (Scomber japonicus) expresses two kiss (kiss1 and kiss2) and three gnrh (gnrh1, gnrh2, and gnrh3) forms in the brain. In the present study, we analyzed expression changes of kiss and gnrh mRNAs in the brain and corresponding GnRH peptides in the brain and pituitary during final ovarian maturation (FOM) and ovulation. METHODS: Female fish possessing late vitellogenic oocytes were injected with GnRH analogue to induce FOM and ovulation. Fish were observed for daily spawning activities and sampled one week post-injection at germinal vesicle migration (GVM), oocyte hydration, ovulation, and post-ovulatory time periods. Changes in relative mRNA levels of kiss and gnrh forms in the brain were determined using quantitative real-time PCR. Changes in GnRH peptides in the brain and pituitary were analyzed using time-resolved fluoroimmunoassay. RESULTS: Both kiss1 and kiss2 mRNA levels in the brain were low at late vitellogenic stage and increased significantly during the GVM period. However, kiss1 mRNA levels decreased during oocyte hydration before increasing again at ovulatory and post-ovulatory periods. In contrast, kiss2 mRNA levels decreased at ovulatory and post-ovulatory periods. Levels of gnrh1 mRNA in the brain increased only during post-ovulatory period. However, levels of gnrh2 and gnrh3 mRNAs were elevated during GVM and then, decreased during oocyte hydration before increasing again at ovulatory period. During post-ovulatory period, both gnrh2 and gnrh3 mRNA levels declined. Peptide levels of all three GnRH forms in the brain were elevated during GVM and oocyte hydration; their levels were significantly lower during late vitellogenic, ovulatory, and post-ovulatory periods. In contrast, pituitary GnRH peptide levels did not show any significant fluctuations, with the GnRH1 peptide levels being many-fold higher than the GnRH2 and GnRH3 forms. CONCLUSION: The results indicate increased expression of multiple Kiss and GnRH forms in the brain and suggest their possible involvement in the regulation of FOM and ovulation in captive female chub mackerel.

Changes in the expression of pituitary gonadotropin subunits during reproductive cycle of multiple spawning female chub mackerel Scomber japonicus.

The endocrine regulation of reproduction in a multiple spawning fish with an asynchronous-type ovary remains largely unknown. The objectives of this study were to monitor changes in the mRNA expression of three gonadotropin (GtH) subunits (GPα, FSHß, and LHß) during the reproductive cycle of the female chub mackerel Scomber japonicus. Cloning and subsequent sequence analysis revealed that the cDNAs of chub mackerel GPα, FSHß, and LHß were 658, 535, and 599 nucleotides in length and encoded 117, 115, and 147 amino acids, respectively. We applied a quantitative real-time PCR assay to quantify the mRNA expression levels of these GtH subunits. During the seasonal reproductive cycle, FSHß mRNA levels remained high during the vitellogenic stages, while GPα and LHß mRNA levels peaked at the end of vitellogenesis. The expression of all three GtH subunits decreased during the post-spawning period. These results suggest that follicle-stimulating hormone (FSH) is involved in vitellogenesis, while luteinizing hormone (LH) functions during final oocyte maturation (FOM). Both GPα and FSHß mRNA levels remained high during the FOM stages of the spawning cycle and increased further just after spawning. Thus, FSH synthesis may be strongly activated just after spawning to accelerate vitellogenesis in preparation for the next spawning. Alternatively, LHß mRNA levels declined during hydration and then increased after ovulation. This study demonstrates that chub mackerel are a good model for investigating GtH functions in multiple spawning fish.

Molecular characterization, tissue distribution, and mRNA expression profiles of two Kiss genes in the adult male and female chub mackerel (Scomber japonicus) during different gonadal stages.

Kisspeptins, encoded by the Kiss1 gene, have emerged as key modulators of reproduction in mammals. In contrast to the placental mammals, some teleosts express two Kiss genes, Kiss1 and Kiss2. In the present study, full-length cDNAs of Kiss1 and Kiss2 in the chub mackerel were cloned and sequenced. Chub mackerel Kiss1 and Kiss2 cDNAs encode 105 and 123 amino acids, respectively. A comparison of the deduced amino acid sequences of chub mackerel Kiss1 and Kiss2 with those of other vertebrate species showed a high degree of conservation only in the kisspeptin-10 region (Kp-10). The Kp-10 of chub mackerel Kiss1 (YNFNSFGLRY) and Kiss2 (FNFNPFGLRF) showed variations at three amino acids. Tissue distribution analysis using quantitative real-time PCR (qRT-PCR) revealed that the Kiss1 and Kiss2 transcripts were expressed in different tissues of adult chub mackerel. In addition, their levels in the adipose tissue exhibited sexually dimorphic expression. Further, to have a basic understanding on the involvement of Kiss1 and Kiss2 in the seasonal gonadal development, their relative mRNA expression profiles in the brain, pituitary, and gonads at different gonadal stages were analyzed using qRT-PCR. Kiss1 and Kiss2 levels in the brain showed a differential expression profile between male and female fish. In males, Kiss1 and Kiss2 levels gradually decreased from the immature stage to spermiation and reached a minimal level during the post-spawning period. In contrast, Kiss1 levels in the brain of females did not vary significantly among the different gonadal stages. However, Kiss2 levels fluctuated as that of males, gradually declining from the immature stage to the post-spawning period. The pituitary Kiss1 levels did not show significant fluctuations. However, Kiss1 levels in the gonads were highly elevated during spermiation and late vitellogenesis compared to the immature and post-spawning period. These results suggest the possible involvement of two Kiss genes in the brain and Kiss1 in the gonads of chub mackerel during seasonal gonadal development.

Immunological characterization and distribution of three GnRH forms in the brain and pituitary gland of chub mackerel (Scomber japonicus).

The presence of three gonadotropin-releasing hormone (GnRH) forms in the brain of the chub mackerel, Scomber japonicus, namely, salmon GnRH (sGnRH), chicken GnRH-II (cGnRH-II), and seabream GnRH (sbGnRH), was confirmed by combined high performance liquid chromatography (HPLC) and time-resolved fluoroimmunoassay (TR-FIA). Immunocytochemical localization of the three GnRH forms in the brain was Investigated by using specific antisera, to elucidate possible roles of each GnRH form in reproduction in this species, and double immunolabeling was used to localize GnRH-ir (immunoreactive) fibers Innervating the pituitary. sGnRH-ir neurons were localized in the ventral olfactory bulb and terminal nerve ganglion region. Further, sGnRH-ir fibers were found in different regions of the brain, with prominent fibers running in parallel in the preoptic area (POA) without entering the pituitary. cGnRH-II-ir cell bodies were observed only in the midbrain tegmentum region, with a wide distribution of fibers, which were dense in the midbrain tegmentum and spinal cord. SbGnRH-ir cell bodies were localized in the nucleus preopticus of the POA, with fibers in the olfactory bulb, POA, and hypothalamus. Among the three GnRH forms, only SbGnRH-ir fibers innervated the pituitary gland from the preoptic-hypothalamic region, targeting follicle stimulating hormone (FSH) and luteinizing hormone (LH)-producing cells in the proximal pars distalis, as demonstrated by double immunocytochemistry. The localization of the GnRH-ir system was similar in male and female fish. These results demonstrate that multiple GnRH forms exist in the brain of the chub mackerel and suggest that they serve different functions, with SbGnRH having a significant role in reproduction in stimulating FSH- and LH-producing cells, and sGnRH and cGnRH-II serving as neurotransmitters or neuromodulators.