SDF-1/CXCR4 signal is involved in the induction of Primordial Germ Cell migration in a model marine fish, Japanese anchovy (Engraulis japonicus).

Primordial germ cells (PGCs) are pivotal for gonadal development and reproductive success. Though artificial induction of sterility by targeting PGCs are gaining popularity due to its advantages in fish surrogacy and biodiversity management, it is often skill and time intensive. In this study, we have focused on understanding the role of PGCs and the chemotactic SDF-1/CXCR4 signaling on gonad development of Japanese anchovy (JA, Engraulis japonicus), an upcoming marine model organism with eco-commercial values, with an aim to develop a novel, easy, and versatile gonad sterilization method. Our data showed that PGC migration related genes, i.e., sdf-1a, sdf-1b, cxcr4a, cxcr4b and vasa, are phylogenetically closer relatives of respective herring (Clupea harengus) and zebrafish (Danio rerio) homolog. Subsequently, PGC marking and live tracing experiments confirmed that PGC migration in JA initiates from 16 hours post fertilization (hpf) followed by PGC settlement in the gonadal ridge at 44 hpf. We found that overexpression of zebrafish sdf-1a mRNA in the germ cell suppresses cxcr4a and increases cxcr4b transcription at 8 hpf, dose dependently disrupts PGC migration at 24-48 hpf, induces PGC death and upregulates sdf-1b at 5 days post hatching. 48 h of immersion treatment with CXCR4 antagonist (AMD3100, Abcam) also accelerated PGC mismigration and pushed the PGC away from gonadal ridge in a dose responsive manner, and further when grown to adulthood caused germ cell less gonad formation in some individuals. Cumulatively, our data, for the first time, suggests that JA PGC migration is largely regulated by SDF1/CXCR4 signaling, and modulation of this signaling has strong potential for sterile, germ cell less gonad preparation at a mass scale. However, further in-depth analysis is pertinent to apply this methodology in marine fish species to successfully catapult Japanese anchovy into a true marine fish model.

Effects of the antifouling agent tributyltin on the sinking behavior, photosynthetic rate and biochemical composition of the marine planktonic diatom Thalassiosira pseudonana.

This study investigated the changes in the sinking rates and physiochemical characteristics of the planktonic marine diatom, Thalassiosira pseudonana, caused by 72 h exposure to antifouling agent tributyltin (TBT) at 1.0 µg L-1 (72-h 10% effective concentration for growth rate, EC10), and 1.7 µg L-1 (EC50). After 72 h of exposure, the sinking rates of T. pseudonana cells were changed from 0.13-0.08 m day-1 in the control, 0.08-0.05 m day-1 in the EC10 treatment, and 0.04-0.006 m day-1 in the EC50 treatment. The results revealed that the sinking rate of T. pseudonana decreased significantly compared with the control at 48 h in the EC10 treatment group and at 24, 48, and 72 h in the EC50 treatment group. The photosynthetic performance index on an absorption basis and the maximum quantum yields of photosystem II also decreased significantly (P < 0.05) in the TBT treatments compared with the control. There was a significant (P < 0.05) positive correlation between sinking rates and cellular protein contents (ng cell-1). Changes in the biochemical and physiochemical composition of the cells suggest that interference with photosynthetic processes by TBT may have reduced their specific gravity and thereby caused a decrease in the sinking rates of T. pseudonana. The results of this investigation suggest the importance of considering the effects of pollutants on the sinking behaviors of diatoms when evaluating the adverse effects of pollutants on marine primary production.

Oxidative Stress Causes Masculinization of Genetically Female Medaka Without Elevating Cortisol.

Medaka (Oryzias latipes) is a teleost fish with an XX/XY sex determination system. Sex reversal from female-to-male (masculinization of XX fish) can be induced through cortisol elevation from exposure to environmental stress such as high temperature during sexual differentiation. However, the effects of oxidative stress, generated via metabolic reactions and biological defense mechanisms, on the sexual differentiation of medaka are unclear. Here, we investigated the effect of oxidative stress on medaka sexual differentiation using hydrogen peroxide (H2O2), which induces oxidative stress in vertebrates. H2O2 treatment from 0 to 5 days post-hatching induced masculinization of wild-type XX medaka, but not of gonadal soma-derived growth factor (gsdf) or peroxisome proliferator-activated receptor alpha-a (pparaa) knockout XX fish. Co-treatment with an oxidative stress inhibitor caused masculinization recovery but co-treatment with a cortisol synthesis inhibitor did not. H2O2 treatment significantly upregulated gsdf and pparaa expression in XX medaka. However, H2O2 did not elevate cortisol levels in medaka larvae during sexual differentiation. These results strongly indicate that oxidative stress induces masculinization of XX medaka without causing elevation of cortisol.

Roles of Gonadotropin Receptors in Sexual Development of Medaka.

The gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), are secreted from the pituitary and bind to the FSH receptor (FSHR) and LH receptor (LHR) to regulate gonadal development in vertebrates. Previously, using fshr-knockout (KO) medaka (Oryzias latipes), we demonstrated that FSH regulates ovarian development by elevating estrogen levels. However, the lhr-KO phenotype in medaka is poorly characterized. Here, we generated lhr-KO medaka using the transcription activator-like effector nuclease (TALEN) technique. We analyzed its phenotype and that of fshr-KO, lhr;fshr double-heterozygotes (double-hetero), and double-KO fish. All genetically male medaka displayed normal testes and were fertile, whereas fshr-KO and double-KO genetically female fish displayed small ovaries containing many early pre-vitellogenic oocytes and were infertile. Although lhr-KO genetically female fish had normal ovaries with full-grown oocytes, ovulation did not occur. Levels of 17α,20ß-dihydroxy-4-pregnen-3-one, which is required for meiotic maturation of oocytes and sperm maturation in teleost fish, were significantly decreased in all KO female medaka ovaries except for double-heteros. Further, 17ß-estradiol levels in fshr-KO and double-KO ovaries were significantly lower than those in double-heteros. These findings indicate that LH is necessary for oocyte maturation and FSH is necessary for follicle development, but that neither are essential for spermatogenesis in medaka.

Recent Progress in the Study of Peroxiredoxin in the Harmful Algal Bloom Species Chattonella marina.

Peroxiredoxin (Prx) is a relatively recently discovered antioxidant enzyme family that scavenges peroxides and is known to be present in organisms from biological taxa ranging from bacteria to multicellular eukaryotes, including photosynthetic organisms. Although there have been many studies of the Prx family in higher plants, green algae, and cyanobacteria, few studies have concerned raphidophytes and dinoflagellates, which are among the eukaryotic algae that cause harmful algal blooms (HABs). In our proteomic study using 2-D electrophoresis, we found a highly expressed 2-Cys peroxiredoxin (2-CysPrx) in the raphidophyte Chattonella marina var. antiqua, a species that induces mass mortality of aquacultured fish. The abundance of the C. marina 2-CysPrx enzyme was highest in the exponential growth phase, during which photosynthetic activity was high, and it then decreased by about a factor of two during the late stationary growth phase. This pattern suggested that 2-CysPrx is a key enzyme involved in the maintenance of high photosynthesis activity. In addition, the fact that the depression of photosynthesis by excessively high irradiance was more severe in the 2-CysPrx low-expression strain (wild type) than in the normal-expression strain (wild type) of C. marina suggested that 2-CysPrx played a critical role in protecting the cell from oxidative stress caused by exposure to excessively high irradiance. In the field of HAB research, estimates of growth potential have been desired to predict the population dynamics of HABs for mitigating damage to fisheries. Therefore, omics approaches have recently begun to be applied to elucidate the physiology of the growth of HAB species. In this review, we describe the progress we have made using a molecular physiological approach to identify the roles of 2-CysPrx and other antioxidant enzymes in mitigating environmental stress associated with strong light and high temperatures and resultant oxidative stress. We also describe results of a survey of expressed Prx genes and their growth-phase-dependent behavior in C. marina using RNA-seq analysis. Finally, we speculate about the function of these genes and the ecological significance of 2-CysPrx, such as its involvement in circadian rhythms and the toxicity of C. marina to fish.

Correlation between Polycyclic Aromatic Hydrocarbons in Wharf Roach (Ligia spp.) and Environmental Components of the Intertidal and Supralittoral Zone along the Japanese Coast.

Polycyclic aromatic hydrocarbon (PAH) concentrations in wharf roach (Ligia spp.), as an environmental indicator, and in environmental components of the intertidal and supralittoral zones were determined, and the PAH exposure pathways in wharf roach were estimated. Wharf roaches, mussels, and environmental media (water, soil and sand, and drifting seaweed) were collected from 12 sites in Japan along coastal areas of the Sea of Japan. PAH concentrations in wharf roaches were higher than those in mussels (median total of 15 PAHs: 48.5 and 39.9 ng/g-dry weight (dw), respectively) except for samples from Ishikawa (wharf roach: 47.9 ng/g-dw; mussel: 132 ng/g-dw). The highest total PAH concentration in wharf roach was from Akita (96.0 ng/g-dw), followed by a sample from Niigata (85.2 ng/g-dw). Diagnostic ratio analysis showed that nearly all PAHs in soil and sand were of petrogenic origin. Based on a correlation analysis of PAH concentrations between wharf roach and the environmental components, wharf roach exposure to three- and four-ring PAHs was likely from food (drifting seaweed) and from soil and sand, whereas exposure to four- and five-ring PAHs was from several environmental components. These findings suggest that the wharf roach can be used to monitor PAH pollution in the supralittoral zone and in the intertidal zone.

Transcriptional Regulation of Müllerian Inhibiting Substance (MIS) and Establishment of a Gonadal Somatic Cell Line Using mis-GFP Transgenic Medaka (Oryzias latipes).

In vertebrate germ cell differentiation, gonadal somatic cells and germ cells are closely related. By analyzing this relationship, it has recently been reported in mammals that primordial germ cells (PGCs), induced from pluripotent stem cells and germline stem cells, can differentiate into functional gametes when co-cultured in vitro with fetal gonadal somatic cells. In some fish species, differentiation into functional sperm by reaggregation or co-culture of gonadal somatic cells and germ cells has also been reported; however, the relationship between gonadal somatic cells and germ cells in these species is not well-understood. Here, we report the transcriptional regulation of Müllerian inhibiting substance (MIS) and the establishment of a gonadal somatic cell line using mis-GFP transgenic fish, in medaka (Oryzias latipes)-a fish model which offers many advantages for molecular genetics. MIS is a glycoprotein belonging to the transforming growth factor ß superfamily. In medaka, mis mRNA is expressed in gonadal somatic cells of both sexes before sex differentiation, and MIS regulates the proliferation of germ cells during this period. Using luciferase assays, we found that steroidogenic factor 1 (SF1) and liver receptor homolog 1 (LRH1) activate medaka mis gene transcription, probably by binding to the mis promoter. We also report that mis-GFP transgenic medaka emit GFP fluorescence specific to gonadal somatic cells in the gonads. By fusing Sertoli cells from transgenic medaka with a cell line derived from medaka hepatoma cancer, we produced a hybridoma cell line that expresses gonadal somatic cell-specific markers, including Sertoli and Leydig cell markers. Moreover, embryonic PGCs co-cultured with the established hybridoma, as feeder cells, proliferated and formed significant colonies after 1 week. PGCs cultured for 3 weeks expressed a germ cell marker dnd, as well as the meiotic markers sycp1 and sycp3. Thus, we here provide the first evidence in teleosts that we have successfully established a gonadal somatic cell-derived hybridoma that can induce both the proliferation and meiosis of germ cells.

Peroxisome proliferator-activated receptor alpha is involved in the temperature-induced sex differentiation of a vertebrate.

Medaka (Oryzias latipes) is a teleost fish with an XX/XY sex determination system, similar to that of mammals. However, under high temperature conditions, XX medaka is masculinised by elevation of cortisol, the major teleost glucocorticoid. In this study, to identify novel factors in the gonads acting downstream from cortisol during sexual differentiation, we performed RNA sequencing (RNA-seq) analysis using the gonadal regions of larvae reared at normal temperature with and without cortisol, and at high temperature. The RNA-seq and real-time PCR analyses showed that expression of some peroxisome proliferator-activated receptor α (PPARα) signalling-targeted genes was increased by cortisol. PPARα agonist treatment induced masculinisation of XX medaka in some cases, and co-treatment of the agonist with cortisol further induced masculinisation, whereas treatment of pparaa knockout medaka with cortisol or the agonist did not induce masculinisation. This study provides the first evidence that PPARα is involved in environmental sex determination in vertebrates.

Time-series responses in photosynthetic activity, 2-cysteine peroxiredoxin gene expression, and proteomics of Chattonella marina var. antiqua under different oxidative stress.

Chattonella spp. are known to produce large amounts of reactive oxygen species (ROS); however, little is known about the mechanisms involved in mitigating the intracellular accumulation of ROS. In this study, a time-series of biological responses in C. marina var. antiqua under different oxidative stress conditions, induced by adding H2O2 at the initial concentrations of 100 and 500 µM, was investigated. Although the added exogenous H2O2 was rapidly consumed at 3 h post-exposure (hpe), intracellular ROS levels were enhanced in the 500 µM H2O2 group but decreased in the 100 µM H2O2 group. Accompanied by increased intracellular ROS levels, the photosynthetic activity of C. marina var. antiqua was considerably inhibited in the 500 µM H2O2 group, but not in the 100 µM H2O2 group. The Fv/Fm ratio and PIABS were negatively correlated with the intracellular ROS level, while the ABS/RC, TR0/RC, and DI0/RC were positively correlated with the intracellular ROS level. Expression of the gene encoding 2-cysteine peroxiredoxin (2-Cys Prx) was up-regulated in 100 µM H2O2 group at 6 hpe, but was down-regulated in 100 µM H2O2 group at 3 and 6 hpe. A negative relationship between the 2-Cys Prx transcript levels and intracellular ROS levels was detected. Results of the 2-DE proteomic analysis confirmed that the 500 µM H2O2 treatment down-regulated the expression of 2-Cys Prx and induced more damage to photosynthetic abilities of C. marina var. antiqua.

Effects of water temperature and light intensity on the acute toxicity of herbicide thiobencarb to a green alga, Raphidocelis subcapitata.

The present study investigated how principal environmental factors such as temperature and light intensity change the toxicological properties of thiobencarb (TB) herbicide to the green alga, Raphidocelis subcapitata. At first, we investigated the inhibitory effect of TB (0, 15.6, 31.2, 62.4, and 125 µg L-1) on growth of R. subcapitata at five temperatures (10, 15, 20, 25, or 30 °C) for 144 h exposure and calculated 72- and 144-h effective concentration values (EC10, 20, and EC50) for growth rate. All EC values significantly decreased with an increasing temperature. The maximum quantum yield of photosystem II in R. subcapitata exposed to 125 µg L-1 of TB was also significantly inhibited with increased temperature. These physiological effects could explain the lower EC values at high temperatures. Then, single and interactive effects of TB, temperature, and light intensity on growth rate were investigated by three-way of analysis of variance. As a result, single and interactive effects were detected in all explanatory variables. These results suggest that temperature and light intensity change the acute toxicity parameter in R. subcapitata exposed to TB and must be considered in evaluating the risk of TB.

Diuron causes sinking retardation and physiochemical alteration in marine diatoms Thalassiosira pseudonana and Skeletonema marinoi-dohrnii complex.

The present research investigated the effect of diuron on sinking rate and the physiochemical changes in two marine diatoms, Thalassiosira pseudonana (single-celled species) and Skeletonema marinoi-dohrnii complex (chain-forming species). The results revealed that the sinking rate of both diatoms exposed to diuron at a level of 50% effective concentration for growth (EC50) decreased significantly compared with the control. Photosynthetic performance (Fv/Fm and PIABS) of both diatoms also decreased significantly with diuron exposure. The number of cells per chain in S. marinoi-dohrnii decreased significantly with diuron treatment, but T. pseudonana cell diameter remained stable. Neutral lipid concentration per cell was significantly higher compared with control at 72 h in both diatom species exposed to EC50 level diuron. And water-soluble protein concentration per cell at 72 h was lower than control in the T. pseudonana EC50 group only. These biochemical changes may decrease specific gravity of cells and seems to cause a decreased sinking rate in diatoms. The positive significant correlation between the numbers of cells per chain and sinking rate in S. marinoi-dohrnii indicated that chain length is also an important factor in sinking rate regulation for chain-forming diatoms. Thus, our present study suggested that suppression of photosynthetic performance and the resultant physiochemical changes induced the decreased sinking rate that may inhibit the normal survival strategy (avoidance from the surface layer where strong light either causes photo-inhibition or interrupts resting cell formation). Therefore, the use of antifouling agents should be considered for the sustainable marine environment.