Wanshu Hong

Review of captive bred species and fry production of marine fish in China.

The artificial breeding of marine fish in China has been developed over a period of more than 50 years. Both species diversity and fry production have greatly increased since the 1980s. By the year 2000, at least 52 species belonging to 24 families of marine fish had been successfully bred. Large quantities of cultured fry can meet the needs of both sea cage culture and pond culture for most species. Fish fry production has entered a period of sustainable development. The number of artificially produced fry of large yellow croaker (Pseudosciaena crocea) exceeded 1.3 billion in year 2000. The species for which more than 10 million fry is produced annually through aquaculture are: red drum (Sciaenops ocellatus), Japanese sea perch (Lateolabrax japonicus), redeye mullet (Liza haematocheila), cuneate drum (Nibea miichthioides), red seabream (Pagrosomus major), milkfish (Chanos chanos) and three-banded sweetlip (Plectorhynchus cinctus). Several millions of artificial fry are cultured annually for the following species: Japanese flounder (Paralichthys olivaceus), black porgy (Sparus macrocephalus), silver sea perch (Lates calcarifer), Russells snapper (Lutjanus russelli), javelin grunt (Pomadasys hasta), miiuy croaker (Miichthys miiuy), Chinese black sleeper (Bostrichthys sinensis) and skewband grunt (Hapalogenys nitens). Fishes belonging to the family Sciaenidae are now the major species for artificial propagation.

Mudskipper genomes provide insights into the terrestrial adaptation of amphibious fishes

Mudskippers are amphibious fishes that have developed morphological and physiological adaptations to match their unique lifestyles. Here we perform whole-genome sequencing of four representative mudskippers to elucidate the molecular mechanisms underlying these adaptations. We discover an expansion of innate immune system genes in the mudskippers that may provide defence against terrestrial pathogens. Several genes of the ammonia excretion pathway in the gills have experienced positive selection, suggesting their important roles in mudskippers’ tolerance to environmental ammonia. Some vision-related genes are differentially lost or mutated, illustrating genomic changes associated with aerial vision. Transcriptomic analyses of mudskippers exposed to air highlight regulatory pathways that are up- or down-regulated in response to hypoxia. The present study provides a valuable resource for understanding the molecular mechanisms underlying water-to-land transition of vertebrates.

STUDIES ON INDUCED OVULATION, EMBRYONIC-DEVELOPMENT AND LARVAL REARING OF THE MUDSKIPPER (BOLEOPHTHALMUS-PECTINIROSTRIS)

Abstract Studies on induced ovulation, embryonic development and larval rearing of the mudskipper ( Boleophthalmus pectinirostris ) were conducted in 1986–1988. Ovulation was successfully induced using hormones and pimozide (PIM). Luteinizing hormone releasing hormone (LHRH-A), at a total dosage of 2 μ g/g, and a combination of LHRH-A (0.2 μ g/g) with PIM (20 μ g/g) or LHRH-A (0.4 μ g/g) with human chorionic gonadotropin (HCG) (30 I.U./g) were the most effective in inducing ovulation. Fertilized eggs were obtained through a semi-dry artificial fertilization method. Fertilization rates ranged from 60 to 90% and hatching rates were between 22 and 80%. The optimum temperature and salinity for hatching were 28°C and 15–25‰, respectively. Newly hatched larvae were reared in outdoor cement pools (water temperatures 25–29.5°C and salinity 15–20‰) to 42 days with survival rates of 7.5–43.8% and an average total length of 20 mm. A total of 7000 fry were obtained. Embryonic and post-embryonic developmental characters were described.

Nuclear Progestin Receptor (Pgr) Knockouts in Zebrafish Demonstrate Role for Pgr in Ovulation but Not in Rapid Non-Genomic Steroid Mediated Meiosis Resumption

Progestins, progesterone derivatives, are the most critical signaling steroid for initiating final oocyte maturation (FOM) and ovulation, in order to advance fully-grown immature oocytes to become fertilizable eggs in basal vertebrates. It is well-established that progestin induces FOM at least partly through a membrane receptor and a non-genomic steroid signaling process, which precedes progestin triggered ovulation that is mediated through a nuclear progestin receptor (Pgr) and genomic signaling pathway. To determine whether Pgr plays a role in a non-genomic signaling mechanism during FOM, we knocked out Pgr in zebrafish using transcription activator-like effector nucleases (TALENs) and studied the oocyte maturation phenotypes of Pgr knockouts (Pgr-KOs). Three TALENs-induced mutant lines with different frame shift mutations were generated. Homozygous Pgr-KO female fish were all infertile while no fertility effects were evident in homozygous Pgr-KO males. Oocytes developed and underwent FOM normally in vivo in homozygous Pgr-KO female compared to the wild-type controls, but these mature oocytes were trapped within the follicular cells and failed to ovulate from the ovaries. These oocytes also underwent normal germinal vesicle breakdown (GVBD) and FOM in vitro, but failed to ovulate even after treatment with human chronic gonadotropin (HCG) or progestin (17α,20β-dihydroxyprogesterone or DHP), which typically induce FOM and ovulation in wild-type oocytes. The results indicate that anovulation and infertility in homozygous Pgr-KO female fish was, at least in part, due to a lack of functional Pgr-mediated genomic progestin signaling in the follicular cells adjacent to the oocytes. Our study of Pgr-KO supports previous results that demonstrate a role for Pgr in steroid-dependent genomic signaling pathways leading to ovulation, and the first convincing evidence that Pgr is not essential for initiating non-genomic progestin signaling and triggering of meiosis resumption.

Why does the mudskipper Boleophthalmus pectinirostris form territories in farming ponds

The mudskipper Boleophthalmus pectinirostris maintained territories in farming ponds during the cold season between November and February. There were two types of territory, one was surrounded by mud-walls, and the other was without mud-walls. Both types of territory were entirely covered by shallow water. The area of walled territories was significantly bigger than that of non-walled ones. The nearest neighbour distance of walled territories was significantly shorter than that of non-walled territories. In January, fish surface activity occurred between 1200 and 1500 hours at which time the territorial water temperature reached a maximum, being significantly higher than that of the air, mud surface or deep burrow. The territorial sediments exhibited a significantly greater benthic microalgal biomass (chlorophyll- a and total chlorophyll- a ) as compared to the non-territorial sediments. The present study indicates that B. pectinirostris forms a territory during the cold season to keep a pool of water that encourages diatom growth and enables the fish to engage in surface activity.

Progestin increases the expression of gonadotropins in pituitaries of male Zebrafish

Our previous study showed that the in vivo positive effects of 17α,20β-dihydroxy-4-pregnen-3-one (DHP), the major progestin in zebrafish, on early spermatogenesis was much stronger than the ex vivo ones, which may suggest an effect of DHP on the expression of gonadotropins. In our present study, we first observed that fshb and lhb mRNA levels in the pituitary of male adult zebrafish were greatly inhibited by 3 weeks exposure to 10nM estradiol (E2). However, an additional 24h 100nM DHP exposure not only reversed the E2-induced inhibition, but also significantly increased the expression of fshb and lhb mRNA. These stimulatory effects were also observed in male adult fish without E2 pretreatment, and a time course experiment showed that it took 24h for fshb and 12h for lhb to respond significantly. Because these stimulatory activities were partially antagonized by a nuclear progesterone receptor (Pgr) antagonist mifepristone, we generated a Pgr-knockout (pgr(-/-)) model using the TALEN technique. With and without DHP in vivo treatment, fshb and lhb mRNA levels of pgr(-/-) were significantly lower than those of pgr(+/+) Furthermore, ex vivo treatment of pituitary fragments of pgr(-/-) with DHP stimulated lhb, but not fshb mRNA expression. Results from double-colored fluorescent in situ hybridization showed that pgr mRNA was expressed only in fshb-expressing cells. Taken together, our results indicated that DHP participated in the regulation of neuroendocrine control of reproduction in male zebrafish, and exerted a Pgr-mediated direct stimulatory effect on fshb mRNA at pituitary level.

Cloning and expression of melatonin receptors in the mudskipper Boleophthalmus pectinirostris: their role in synchronizing its semilunar spawning rhythm.

The mudskipper Boleophthalmus pectinirostris, a burrow-dwelling fish inhabiting intertidal mudflats, spawns only once during the spawning season around either the first or last lunar quarters. To understand the molecular mechanisms regulating this semilunar spawning rhythm, we cloned all melatonin receptor subtypes (mtnr1a1.4, mtnr1a1.7, mtnr1b, and mtnr1c). Expression of three melatonin receptor subtypes (except mtnr1c) was found in the ovaries. In contrast, the expression of all receptor subtypes was found in the diencephalon and the pituitary. In the fully-grown follicles, only mtnr1a1.7 mRNA was detected in both the isolated follicle layers and denuded oocytes. Interestingly, the transcript levels of both mtnr1a1.4 in the diencephalon and mtnr1a1.7 in the ovary displayed two cycles within one lunar month, and peaked around the first and last lunar quarters. We used 17α,20β-dihydroxy-4-pregnen-3-one (DHP), a maturation-inducing hormone, as a biomarker to examine the involvement of melatonin receptors in the control of the spawning cycle. Melatonin significantly increased the plasma DHP level 1h post intraperitoneal injection. Melatonin also directly stimulated ovarian fragments in vitro to produce a significantly higher amount of DHP. Taken together, these results provided the first evidence that melatonin receptors were involved in the synchronization of the semilunar spawning rhythm in the female mudskipper by acting through the HPG axis and/or directly on ovarian tissues to stimulate the production of DHP.

Cloning and expression pattern of gsdf during the first maleness reproductive phase in the protandrous Acanthopagrus latus.

Gonadal soma-derived factor (Gsdf) is a new member of the transforming growth factor beta superfamily. As a teleost- and gonad-specific growth factor, several studies indicate that Gsdf plays an important role in early germ cell development. In the present study, for the first time, a 1700-bp long gsdf gene was cloned from a protandrous species, Acanthopagrus latus. We further analyzed the cellular localization and the expression patterns of gsdf in respective testicular and ovarian zones during the first maleness reproductive phase. The results showed that gsdf transcripts were highly expressed in the ovotestis during sex differentiation, and the somatic cells of the testicular zone expressed many more gsdf transcripts than those of the ovarian zone. At the onset of puberty, the gsdf expression levels decreased gradually during spermatogenesis. Conversely, the ovarian zone exhibited a stable increase pattern which was similar to the plasma 17β-estradiol (E2) levels. These results suggested that Gsdf may participates in early germ cell development, e.g. proliferation and differentiation of spermatogonia and oogonia in A. latus.

Complete mitochondrial genome of the mudskipper Boleophthalmus boddarti (Perciformes, Gobiidae).

Abstract The Boddarts goggle-eyed mudskipper, Boleophthalmus boddarti (Perciformes, Gobiidae) is an amphibious fish, inhabiting brackish waters of estuaries and builds burrows in soft mud along the intertidal zone. In this paper, the complete mitochondrial genome sequence of B. boddarti was firstly determined. The circle genome (16,727 bp) comprises 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes and 1 control region. The overall base composition of B. boddarti is 29.1% for C, 28.9% for A, 25.9% for T, and 16.0% for G, with a slight A + T bias of 54.8%. The termination-associated sequence, conserved sequence block domains, and a 131-bp tandem repeat were found in the control region. It has the typical vertebrate mitochondrial gene arrangement.

Cloning and olfactory expression of progestin receptors in the Chinese black sleeper Bostrichthys sinensis.

Our previous studies suggested that 17α,20β-dihydroxy-4-pregnen-3-one (DHP), an oocyte maturation inducing progestin, also acts as a sex pheromone in Chinese black sleeper Bostrichthys sinensis, a fish species that inhabits intertidal zones and mates and spawns inside a muddy burrow. The electro-olfactogram response to DHP increased during the breeding season. In the present study, we cloned the cDNAs of the nine progestin receptors (pgr, paqr5, 6, 7(a, b), 8, 9, pgrmc1, 2) from B. sinensis, analyzed their tissue distribution, and determined the expression in the olfactory rosette during the reproductive cycle in female and male fish. The deduced amino acid sequences of the nine progestin receptors share high sequence identities with those of other fish species and relatively lower homology with their mammalian counterparts, and phylogenetic analyses classified the nine B. sinensis progestin receptors into their respective progestin receptor groups. Tissue distribution of B. sinensis progestin receptors showed differential expression patterns, but all these nine genes were expressed in the olfactory rosette. Interestingly, paqr5 mRNA was found in the intermediate and basal parts of the olfactory epithelium but not in the central core using in situ hybridization, and its expression level was the highest in the olfactory rosette among the tissues examined. These results suggested Paqr5 may have an important role for transmitting progestin signaling in the olfactory system. The expression levels of paqr7a and paqr7b, pgr and pgrmc2 mRNA peaked around the mid meiotic stage, and that of paqr8 peaked at late meiotic stage in the olfactory rosette in males, while the olfactory expression of paqr5 decreased gradually as spermatogenesis progressed. In contrast, the expression of the progestin receptors did not change significantly during the development of the ovary in the olfactory rosette in females, except that of pgr. Interestingly, the changes of paqr8 expression in the olfactory rosette in males mirrored the changes of plasma DHP levels in females during the reproductive cycle, suggesting the Paqr8 may also be important for deciphering progestin signaling released by female. To our knowledge, this is the first time to demonstrate the presence of all known progestin receptors in a teleost olfactory rosette, and to show different expressions between the males and females during the reproductive cycle. This study provides the first evidence on changes of all purported progestin receptors during a reproductive cycle in teleost olfactory rosette, and suggests that distinct olfactory sensitivities to DHP may be due to the changes and compositions of each progestin receptor in B. sinensis.