Two pathways regulate insulin-like growth factor genes in the brain and liver of the tropical damselfish Chrysiptera cyanea: A possible role for melatonin in the actions of growth and thyroid hormones.

External and internal factors are involved in controlling the growth of fishes. However, little is known about the mechanisms by which external factors trigger stimulus signals. This study explored the physiological roles of melatonin in the transcription of growth-related genes in the brain and liver of Chrysiptera cyanea, a tropical damselfish with long-day preference. In brain samples of this species collected at 4-h intervals, the transcript levels of arylalkylamine N-acetyltransferase2 (aanat2), the rate-limiting enzyme of melatonin synthesis, and growth hormone (gh) peaked at 20:00 and 00:00, respectively. Concomitantly, the transcript levels of insulin-like growth factors (igf1 and igf2) in the brain and liver were upregulated during the scotophase. Levels of iodothyronine deiodinases (dio2 and dio3), enzymes that convert thyroxine (T4) to triiodothyronine (T3) and reverse T3, respectively, increased in the brain (dio2 and dio3) and liver (dio2) during the photophase, whereas dio3 levels in the liver showed the opposite trend. Fish reared in melatonin-containing water exhibited significant increases in the transcription levels of gh and igf1 in the brain and igf1 in the liver, suggesting that growth in this fish is positively regulated by the GH/IGF pathway on a daily basis. Melatonin treatment also stimulated the transcript levels of dio2 and dio3 in the liver, but not in the brain. Fish consuming pellets containing T3, but not T4, showed significant increases in gh and igf1 in the brain and igf1 and igf2 in the liver, suggesting that the intercellular actions of the TH/IGF pathway have an impact on growth on a daily basis. In summary, IGF synthesis and action in the brain and liver undergo dual regulation by distinct hormone networks, which may also be affected by daily, seasonal, or nutritional factors.

Effects of dopamine and melatonin treatment on the expression of the genes associated with artificially induced sexual maturation in Japanese eel, Anguilla japonica.

Japanese eel (Anguilla japonica) is a commercially important fish species in Asia. Understanding factors like photoperiod, temperature, and lunar cycles is crucial for successful aquaculture and managing its reproduction. Melatonin and dopamine (DA) are essential for regulating reproduction in vertebrates, including fish. This study investigated the effects of melatonin and DA on the reproductive system of mature male Japanese eels to better understand reproductive regulation in fish. To clarify the effects of these hormones on sexual maturation in eels, a critical stage in the reproductive process, sexual maturation was induced by injecting human chorionic gonadotropin, which stimulates the production of sex hormones. To check the effect of melatonin and DA on sexual maturation, DA, melatonin, and DA + domperidone were intraperitoneally injected into fish from each group (six per treatment) at a dose of 1 mg/kg body weight. The fish were then examined using quantitative RT-PCR by comparing the messenger RNA level of reproduction-related genes (gonadotropin releasing hormone 1; gnrh1, gonadotropin releasing hormone 2; gnrh2, follicle stimulating hormone; fshß, luteinizing hormone; lhß and DA receptor 2b; d2b), involved in the gonadotropic axis in eels, to those that received a control injection. The results indicate significant differences in the expression levels of gnrh1, gnrh2 and d2b in the brain and d2b, fshß, lhß in the pituitary at different stages of sexual maturation. Melatonin appears to enhance the production of sex gonadotropins, whereas DA inhibits them. These findings suggest an interaction between melatonin and DA in regulating reproduction in Japanese eels.

DNA barcoding and morphological identification of spiny lobsters in South Korean waters: a new record of Panulirus longipes and Panulirus homarus homarus.

To date, 19 species of spiny lobsters from the genus Panulirus have been discovered, of which only P. japonicus, P. penicilatus, P. stimpsoni, and P. versicolor have been documented in South Korean waters. In this study, we aimed to identify and update the current list of spiny lobster species that inhabit South Korean waters based on the morphological features and the phylogenetic profile of cytochrome oxidase I (COI) of mitochondrial DNA (mtDNA). Spiny lobsters were collected from the southern and eastern coasts of Jeju Island, South Korea. Phylogenetic analyses were performed using neighbor-joining (NJ), maximum likelihood (ML), and Bayesian inference (BI) methods. The ML tree was used to determine the spiny lobster lineages, thereby clustering the 17 specimens collected in this study into clades A, B, C, and D, which were reciprocally monophyletic with P. japonicus, P. homarus homarus, P. longipes, and P. stimpsoni, respectively. These clades were also supported by morphological examinations. Interestingly, morphological variations, including the connected pleural and transverse groove at the third abdominal somite, were observed in four specimens that were genetically confirmed as P. japonicus. This finding is novel within the P. japonicus taxonomical reports. Additionally, this study updates the documentation of spiny lobsters inhabiting South Korean waters as P. longipes and P. homarus homarus were recorded for the first time in this region.

Effect of short- and long-term melatonin treatments on the reproductive activity of the tropical damselfish Chrysiptera cyanea.

Photoperiod plays a role in controlling the initiation and termination of reproduction in fish. Melatonin is an internal transducer of environmental photoperiod and is involved in regulating reproduction. The present study aimed to examine how melatonin impacts the transcript levels of kisspeptin (kiss1 and kiss2), gonadotropin-releasing hormones (gnrh1), and the ß-subunit of gonadotropins (fshß and lhß) in the brain of the sapphire devil, a tropical damselfish with long photoperiod preference. Feeding mature females with melatonin-containing pellets inhibited increases in the transcript levels of kiss1, gnrh1, and lhß within 3 h. Continuous melatonin treatment for 1 week resulted in oocyte regression and downregulation of kiss2, gnrh1, fshß, and lhß. When the transcript levels of kiss1 and gnrh1 were measured at 4-h intervals in the brain of sapphire devil, a day-high/night-low fluctuation was observed. The hypothalamic-pituitary-gonadal (HPG) axis may be influenced by melatonin, exerting a negative effect at night because the transcript levels of aralkylamine N-acetyltransferase (aanat2) increased during the scotophase. The expression of aanat2 was higher under short-day than long-day conditions, suggesting that there is a seasonal change in melatonin levels at night. It was concluded that change in photoperiod becomes a key factor for controlling the hormone synthesis in the HPG axis through melatonin.

Food availability alters expression profiles of genes in relation to reproduction and nutrition in the females of tropical damselfish (Chrysiptera cyanea).

This study evaluated the effects of food availability on the transcript levels of genes related to reproduction and growth in the sapphire devil (Chrysiptera cyanea), a tropical damselfish. Nonbreeding fish were reared at high-food (HF) and low-food (LF) levels for 4 weeks under long-days. Vitellogenic oocytes could be observed in the ovaries of the HF group. The quantitative polymerase chain reaction analysis revealed that lhß and cyp19b in the brains, vtg and igf1 in the livers and cyp19a in the ovaries of HF fish were significantly higher than that of LF fish, suggesting that estradiol-17ß (E2) synthesis in the ovary and brain is activated when suitable permissive factors are available to fish. Food limitation lowered hepatic igf1 and dio2, suggesting that the TH-IGF1 signaling system functions in the liver, and that food availability altered hepatic deiodination activities related to intercellular levels of thyroid hormones. Hepatic dio2 significantly decreased when fish were immersed for 3 days in E2-containing seawater; this suggests that E2 impedes the conversion of T4 to T3 in the liver. Our study shows that igf1 was upregulated in accordance with HF-induced vitellogenesis but downregulated by E2 treatment, suggesting that igf1 is bidirectional and altered by maturational status. Once vitellogenesis begins under a suitable range of proximal factors, fish need to maintain their nutritional status because food availability is a permissive factor for their reproduction.

Molecular cloning of insulin-like growth factor 3 (igf3) and its expression in the tissues of a female damselfish, Chrysiptera cyanea, in relation to seasonal and food-manipulated reproduction.

Food availability is a permissive determinant that drives gonadal activity in fish. The present study aimed to clarify the interactions between reproductive and nutritive statuses in the sapphire devil (Chrysiptera cyanea), a tropical damselfish with a long-day preference for reproduction. Insulin-like growth factor 3 (IGF3), a novel IGF that likely plays a role in gonadal maturation, was closely monitored in the sapphire devil. The cDNA of sapphire devil igf3 had an open reading frame of 443 base pairs (146 amino acid residues). Phylogenetic analyses revealed that sapphire devil IGF3 was clustered within the teleost IGF3 family. The transcript levels of sapphire devil igf3 increased in the brain, liver, and ovary of the fish during the late vitellogenic phase, suggesting that it plays a role in reproduction. Immersion of the fish in seawater containing estradiol-17ß suppressed transcript levels of sapphire devil igf3 in the liver, but not in the brain, suggesting that intensive protein synthesis in relation to vitellogenesis negatively impacts somatic metabolism in this tissue. When fish were reared with high or low food under conditions of photoperiod (LD = 14:10) and temperature (at 25-28 °C) during the non-reproductive season, ovarian development was induced in high-food fish. Furthermore, prior to ovarian development in the high-food fish, the transcript levels of sapphire devil igf3 increased in the brain, liver, and ovary. These results indicated crosstalk between the reproductive and growth networks and suggested that a metabolic shift, from growth mode to reproductive mode, occurs in peripheral tissues when nutritive status is improved under suitable conditions of photoperiod and water temperature.

Expression profiles of types 2 and 3 iodothyronine deiodinase genes in relation to vitellogenesis in a tropical damselfish, Chrysiptera cyanea.

Thyroid hormone (TH) is involved in regulating the reproduction of vertebrates. Its physiological action in the target tissues is due to the conversion of TH by iodothyronine deiodinases. In this study, we aimed to clone and characterize type 2 (sdDio2) and type 3 (sdDio3) of the sapphire devil Chrysiptera cyanea, a tropical damselfish that undergoes active reproduction under long-day conditions, and to study the involvement of THs in the ovarian development of this species. When the cDNAs of sdDio2 and sdDio3 were partially cloned, they had deduced amino acid sequences of lengths 271 and 267, respectively, both of which were characterized by one selenocysteine residue. Real-time quantitative PCR (qPCR) revealed that both genes are highly expressed in the whole brain, and sdDio2 and sdDio3 are highly transcribed in the liver and ovary, respectively. In situ hybridization analyses showed positive signals of sdDio2 and sdDio3 transcripts in the hypothalamic area of the brain. Little change in mRNA abundance of sdDio2 and sdDio3 in the brain was observed during the vitellogenic phases. It is assumed that simultaneous activation and inactivation of THs occur in this area because oral administration of triiodothyronine (T3), but not of thyroxine (T4), upregulated mRNA abundance of both genes in the brain. The transcript levels of sdDio2 in the liver and sdDio3 in the ovary increased as vitellogenesis progressed, suggesting that, through the metabolism of THs, sdDio2 and sdDio3 play a role in vitellogenin synthesis in the liver and yolk accumulation/E2 synthesis in the ovary. Taken together, these results suggest that iodothyronine deiodinases act as a driver for vitellogenesis in tropical damselfish by conversion of THs in certain peripheral tissues.

The mRNA expression patterns of kisspeptins, GnRHs, and gonadotropins in the brain and pituitary gland of a tropical damselfish, Chrysiptera cyanea, during the reproductive cycle.

The sapphire devil (Chrysiptera cyanea) is a tropical damselfish that undergoes active reproduction under long-day conditions. To elucidate the physiological regulation of the brain-pituitary-gonadal axis in female sapphire devil, we cloned and characterized the genes of two kisspeptins (kiss1 and kiss2), three gonadotropin-releasing hormones (gnrh1, gnrh2, gnrh3), and the ß-subunit of two gonadotropins (fshß and lhß) and investigated the gene expression changes during ovarian development. Quantitative polymerase chain reaction analyses in various brain parts revealed high expression levels of kiss1, kiss2, and gnrh2 in the diencephalon; gnrh2 and gnrh3 in the telencephalon; and fshß and lhß in the pituitary. In situ hybridization (ISH) analyses revealed positive signals of kiss1 in the dorsal and ventral habenular nucleus and of kiss2 in the dorsal and ventral parts of the nucleus of the lateral recess. This analysis showed gnrh1 expression in the preoptic area (POA), suggesting that GnRH1 plays a stimulating role in the secretion of gonadotropins from the pituitary of the sapphire devil. High transcription levels of kiss1, kiss2, gnrh1, gnrh2, fshß, and lhß were observed in the brain during the late vitellogenic stage, suggesting their involvement in the physiological processes of vitellogenesis. Immersion of fish in estradiol-17ß (E2)-containing seawater resulted in increased expression of kiss2 and gnrh1 in their brains. This study showed that kiss-expressing neurons in the diencephalon are influenced by E2, leading to upregulation of gnrh1 in the POA and of fshß and lhß in the pituitary during vitellogenesis.

Changes in mRNA abundance of insulin-like growth factors in the brain and liver of a tropical damselfish, Chrysiptera cyanea, in relation to seasonal and food-manipulated reproduction.

Food availability can become a factor driving the reproductive activity of tropical fish, particularly when primary production within their habitats fluctuates with tropical monsoons. The present study examined the involvement of insulin-like growth factors (IGF) in controlling the reproduction of the sapphire devil Chrysiptera cyanea, a reef-associated damselfish that is capable of manipulating its reproductive activity based on food availability. We cloned and characterized the cDNAs of igf1 and igf2 and determined their transcript levels in relation to seasonal and food-manipulated reproduction. The partial cDNAs of sapphire devil igf1 and igf2 had open reading frames (ORFs) composed of 600 bp (155 amino acid residue) and 636 bp (211 aa), respectively. Phylogenetic analyses revealed that IGF1 and IGF2 of the sapphire devil were clustered into those of teleosts. The gonadosomatic index increased from March to June. Vitellogenic oocytes and ovulatory follicles were observed in ovaries from May to June, which suggests that the spawning season lasts for at least 2 months. The hepatosomatic index, but not the condition factor, increased in March and June. The transcript levels of igf1 in the brain, but not in the liver, increased in April, June (vitellogenesis) and July (post vitellogenesis). Ovarian activity during the spawning season was maintained by high food supply (HH) for 30 days, although it was suppressed in the food-restriction treatment (LL) and restored in the re-feeding treatment (LH). The transcript levels of igfs in the brain, but not in the liver, in LH were lower than those in HH and LL. Moreover, immersing fish in seawater containing estradiol-17ß suppressed transcript levels of igfs in the liver, but not in the brain. We conclude that reproductive activity during the spawning season is influenced by nutritive conditions and that crosstalk exists between the reproductive and growth network in the neural and peripheral tissues, thus controlling the reproductive activity of this species.

The importance of standardization for biodiversity comparisons: A case study using autonomous reef monitoring structures (ARMS) and metabarcoding to measure cryptic diversity on Mo'orea coral reefs, French Polynesia.

The advancement of metabarcoding techniques, declining costs of high-throughput sequencing and development of systematic sampling devices, such as autonomous reef monitoring structures (ARMS), have provided the means to gather a vast amount of diversity data from cryptic marine communities. However, such increased capability could also lead to analytical challenges if the methods used to examine these communities across local and global scales are not standardized. Here we compare and assess the underlying biases of four ARMS field processing methods, preservation media, and current bioinformatic pipelines in evaluating diversity from cytochrome c oxidase I metabarcoding data. Illustrating the ability of ARMS-based metabarcoding to capture a wide spectrum of biodiversity, 3,372 OTUs and twenty-eight phyla, including 17 of 33 marine metazoan phyla, were detected from 3 ARMS (2.607 m2 area) collected on coral reefs in Mo'orea, French Polynesia. Significant differences were found between processing and preservation methods, demonstrating the need to standardize methods for biodiversity comparisons. We recommend the use of a standardized protocol (NOAA method) combined with DMSO preservation of tissues for sessile macroorganisms because it gave a more accurate representation of the underlying communities, is cost effective and removes chemical restrictions associated with sample transportation. We found that sequences identified at ≥ 97% similarity increased more than 7-fold (5.1% to 38.6%) using a geographically local barcode inventory, highlighting the importance of local species inventories. Phylogenetic approaches that assign higher taxonomic ranks accrued phylum identification errors (9.7%) due to sparse taxonomic coverage of the understudied cryptic coral reef community in public databases. However, a ≥ 85% sequence identity cut-off provided more accurate results (0.7% errors) and enabled phylum level identifications of 86.3% of the sequence reads. With over 1600 ARMS deployed, standardizing methods and improving databases are imperative to provide unprecedented global baseline assessments of understudied cryptic marine species in a rapidly changing world.