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Estrogenic endocrine disrupting chemicals (EEDC) have been suspected to impact offspring in a transgenerational manner via modifications of the germline epigenome in the directly exposed generations. A holistic assessment of the concentration/ exposure duration-response, threshold level, and critical exposure windows (parental gametogenesis and embryogenesis) for the transgenerational evaluation of reproduction and immune compromise concomitantly will inform the overall EEDC exposure risk. We conducted a multigenerational study using the environmental estrogen, 17α-ethinylestradiol (EE2), and the marine laboratory model fish Oryzias melastigma (adult, F0) and their offspring (F1-F4) to identify transgenerationally altered offspring generations and phenotype persistence. Three exposure scenarios were used: short parental exposure, long parental exposure, and a combined parental and embryonic exposure using two concentrations of EE2 (33ng/L, 113ng/L). The reproductive fitness of fish was evaluated by assessing fecundity, fertilization rate, hatching success, and sex ratio. Immune competence was assessed in adults via a host-resistance assay. EE2 exposure during both parental gametogenesis and embryogenesis was found to induce concentration/ exposure duration-dependent transgenerational reproductive effects in the unexposed F4 offspring. Furthermore, embryonic exposure to 113 ng/L EE2 induced feminization of the directly exposed F1 generation, followed by subsequent masculinization of the F2 and F3 generations. A sex difference was found in the transgenerationally impaired reproductive output with F4 females being sensitive to the lowest concentration of EE2 (33 ng/L) upon long-term ancestral parent exposure (21 days). Conversely, F4 males were affected by ancestral embryonic EE2 exposure. No definitive transgenerational impacts on immune competence were identified in male or female offspring. In combination, these results indicate that EEDCs can be transgenerational toxicants that may negatively impact the reproductive success and population sustainability of fish populations., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Antimicrobial peptides (AMPs) have emerged as a promising alternative to small molecule antibiotics. Although AMPs have previously been isolated in many organisms, efforts on the systematic identification of AMPs in fish have been lagging. Here, we collected peptides from the plasma of medaka (Oryzias latipes) fish. By using mass spectrometry, 6399 unique sequences were identified from the isolated peptides, among which 430 peptides were bioinformatically predicted to be potential AMPs. One of them, a thermostable 13-residue peptide named BING, shows a broad-spectrum toxicity against pathogenic bacteria including drug-resistant strains, at concentrations that presented relatively low toxicity to mammalian cell lines and medaka. Proteomic analysis indicated that BING treatment induced a deregulation of periplasmic peptidyl-prolyl isomerases in gram-negative bacteria. We observed that BING reduced the RNA level of cpxR, an upstream regulator of envelope stress responses. cpxR is known to play a crucial role in the development of antimicrobial resistance, including the regulation of genes involved in drug efflux. BING downregulated the expression of efflux pump components mexB, mexY and oprM in P. aeruginosa and significantly synergised the toxicity of antibiotics towards these bacteria. In addition, exposure to sublethal doses of BING delayed the development of antibiotic resistance. To our knowledge, BING is the first AMP shown to suppress cpxR expression in Gram-negative bacteria. This discovery highlights the cpxR pathway as a potential antimicrobial target.

Hypoxia, a low environmental oxygen level, is a common problem in the ocean globally. Hypoxia has been known to cause disruption to the endocrine system of marine organisms in both laboratory and field studies. Our previous studies have demonstrated the sex-specific response to hypoxia in the neural and reproductive systems of marine fish. In the current report, we aim to study the sex-specific hepatic response of fish at the transcriptome level to hypoxic stress. By using a comparative transcriptome analysis, followed by a systematic bioinformatics analysis including Database for Annotation, Visualization and Integrated Discovery (DAVID) and Ingenuity Pathway Analysis (IPA), we found that hypoxia altered expression of genes related to cell proliferation and apoptosis of hepatocytes, which are associated with human pathologies, such as liver inflammation hepatic steatosis and steatohepatitis. Furthermore, we observed sex-specific responses in the livers of fish through different cell signaling pathways. In female fish, hypoxia causes dysregulation of expression of genes related to impairment in endoplasmic reticulum structure and liver metabolism. In male fish, genes associated with redox homeostasis and fatty acid metabolism were altered by hypoxic stress. The findings of this study support the notion that hypoxia could cause sex-specific changes (hepatic toxicity and changes) in marine fish., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Germ plasm, a cytoplasmic factor of germline cell differentiation, is suggested to be a perspective tool for in vitro meiotic differentiation. To discriminate between the: (1) germ plasm-related structures (GPRS) involved in meiosis triggering; and (2) GPRS involved in the germ plasm storage phase, we investigated gametogenesis in the marine medaka Oryzias melastigma. The GPRS of the mitosis-to-meiosis period are similar in males and females. In both sexes, five events typically occur: (1) turning of the primary Vasa-positive germ plasm granules into the Vasa-positive intermitochondrial cement (IMC); (2) aggregation of some mitochondria by IMC followed by arising of mitochondrial clusters; (3) intramitochondrial localization of IMC-originated Vasa; followed by (4) mitochondrial cluster degradation; and (5) intranuclear localization of Vasa followed by this protein entering the nuclei (gonial cells) and synaptonemal complexes (zygotene-pachytene meiotic cells). In post-zygotene/pachytene gametogenesis, the GPRS are sex specific; the Vasa-positive chromatoid bodies are found during spermatogenesis, but oogenesis is characterized by secondary arising of Vasa-positive germ plasm granules followed by secondary formation and degradation of mitochondrial clusters. A complex type of germ plasm generation, 'the follicle cell assigned germ plasm formation', was found in late oogenesis. The mechanisms discovered are recommended to be taken into account for possible reconstruction of those under in vitro conditions.

Medaka ( Oryzias sp.) is an important fish species in ecotoxicology and considered as a model species due to its biological features including small body size and short generation time. Since Japanese medaka Oryzias latipes is a freshwater species with access to an excellent genome resource, the marine medaka Oryzias melastigma is also applicable for the marine ecotoxicology. In genome era, a high-density genetic linkage map is a very useful resource in genomic research, providing a means for comparative genomic analysis and verification of de novo genome assembly. In this study, we developed a high-density genetic linkage map for O. melastigma using restriction-site associated DNA sequencing (RAD-seq). The genetic map consisted of 24 linkage groups with 2,481 single nucleotide polymorphism (SNP) markers. The total map length was 1,784 cM with an average marker space of 0.72 cM. The genetic map was integrated with the reference-assisted chromosome assembly (RACA) of O. melastigma , which anchored 90.7% of the assembled sequence onto the linkage map. The values of complete Benchmarking Universal Single-Copy Orthologs were similar to RACA assembly but N50 (23.74 Mb; total genome length 779.4 Mb; gap 5.29%) increased to 29.99 Mb (total genome length 778.7 Mb; gap 5.2%). Using MapQTL analysis with SNP markers, we identified a major quantitative trait locus for sex traits on the Om10. The integration of the genetic map with the reference genome of marine medaka will serve as a good resource for studies in molecular toxicology, genomics, CRISPR/Cas9, and epigenetics., (Copyright © 2019 Lee et al.)

Perfluorobutanesulfonate (PFBS), an environmental pollutant of emerging concern, significantly impairs offspring development and overall health after parental exposure. However, the true inducer of offspring developmental defects among the complexity of parental influences remains unknown. In the present study, marine medaka ( Oryzias melastigma ) were exposed to environmentally realistic concentrations of PFBS (0, 1, 3, and 10 μg/L) for an entire life cycle. By mixing and mating control and exposed medaka (male or female), a crossbreeding strategy was employed to produce offspring eggs from various crossbreeds, with the aim of differentiating the maternal and paternal influences. Measurements of swimming performance in larval offspring showed that larvae of exposed male parents swam hyperactively in comparison to the control larvae. Contrasting trends in PFBS transfer and maternal factor transfer (e.g., proteins and lipids) to that of swimming behavior eliminated these two factors as major inducers of offspring developmental impairment. Inheritance of the exposed paternal methylome marks in offspring may be partially responsible for abnormal swimming behavior, although different toxic mechanisms may be involved depending on the exposure concentration. Overall, these findings suggest that inheritance of epigenetic modifications implicates a long-lasting threat of PFBS to the fitness and sustainability of fish populations.

Hypoxia is a pressing environmental problem in both marine and freshwater ecosystems globally, and this problem will be further exacerbated by global warming in the coming decades. Recently, we reported that hypoxia can cause transgenerational impairment of sperm quality and quantity in fish (in F0, F1, and F2 generations) through DNA methylome modifications. Here, we provide evidence that female fish ( Oryzias melastigma) exposed to hypoxia exhibit reproductive impairments (follicle atresia and retarded oocyte development), leading to a drastic reduction in hatching success in the F2 generation of the transgenerational group, although they have never been exposed to hypoxia. Further analyses show that the observed transgenerational impairments in ovarian functions are related to changes in the DNA methylation and expression pattern of two gene clusters that are closely associated with stress-induced cell cycle arrest and cell apoptosis. The observed epigenetic and transgenerational alterations suggest that hypoxia may pose a significant threat to the sustainability of natural fish populations.

Estrogenic endocrine disrupting chemicals (EEDCs) are present ubiquitously in sediments and aquatic ecosystems worldwide. The detrimental impact of EEDCs on the reproduction of wildlife is widely recognized. Increasing evidence shows the immunosuppressive effects of EEDCs in vertebrates. Yet, no studies have considered concomitantly EEDC-induced impacts on reproductive impairment and immune suppression in vivo, which are deemed essential for risk assessment and environmental monitoring. In this study, EE2 was used as a representative EEDC, for parallel evaluation of EEDC-induced immune suppression (immune marker gene expression, leukocyte numbers, host resistance assay, and immune competence index) and reproductive impairment (estrogen responsive gene expression, fecundity, fertilization success, hatching success, and reproductive competence index) in an established fish model (marine medaka Oryzias melastigma), considering sex-specific induction and adaptation and recovery responses under different EE2 exposure scenarios. The findings in marine medaka reveal distinct sex differences in the EE2-mediated biological responses. For female fish, low concentration of exogenous EE2 (33 ng/L) could induce hormesis (immune enhancement), enable adaptation (restored reproduction) and even boost fish resistance to bacterial challenge after abatement of EE2. However, a prolonged exposure to high levels of EE2 (113 ng/L) not only impaired F0 immune function, but also perturbed females recovering from reproductive impairment, resulting in a persistent impact on the F1 generation output. Thus, for female fish, the exposure concentration of EE2 is more critical than the dose of EE2 in determining the impacts of EE2 on immune function and reproduction. Conversely, male fish are far more sensitive than females to the presence of low levels of exogenous EE2 in water and the EE2-mediated biological impacts are clearly dose-dependent. It is also evident in male fish that direct contact of EE2 is essential to sustain impairments of immune competence and reproductive output as well as deregulation of immune function genes in vivo. The immunomodulatory pathways altered by EE2 were deciphered for male and female fish, separately. Downregulation of hepatic tlr3 and c3 (in female) and tlr3, tlr5 and c3 (in male) may be indicative of impaired fish immune competence. Taken together, impaired immune competence in the EE2-exposed fish poses an immediate thread on the survival of F0 population. Impaired reproduction in the EE2-exposed fish can directly affect F1 output. Parallel evaluation of immune competence and reproduction are important considerations when assessing the risk of sublethal levels of EE2/EEDCs in aquatic environments., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Bone morphogenetic protein (BMP) signaling is essential for osteogenesis. However, recombinant human BMPs (rhBMPs) exhibit large inter-individual variations in local bone formation during clinical spinal fusion. Smurf1 ubiquitinates BMP downstream molecules for degradation. Here, we classify age-related osteoporosis based on distinct intraosseous BMP-2 levels and Smurf1 activity. One major subgroup with a normal BMP-2 level and elevated Smurf1 activity (BMP-2 n /Smurf1 e ) shows poor response to rhBMP-2 during spinal fusion, when compared to another major subgroup with a decreased BMP-2 level and normal Smurf1 activity (BMP-2 d /Smurf1 n ). We screen a chalcone derivative, i.e., 2-(4-cinnamoylphenoxy)acetic acid, which effectively inhibits Smurf1 activity and increases BMP signaling. For BMP-2 n /Smurf1 e mice, the chalcone derivative enhances local bone formation during spinal fusion. After conjugating to an osteoblast-targeting and penetrating oligopeptide (DSS) 6 , the chalcone derivative promotes systemic bone formation in BMP-2 n /Smurf1 e mice. This study demonstrates a precision medicine-based bone anabolic strategy for age-related osteoporosis.

Selenium (Se) is an essential element and its biological activity is related to its speciation. It is also well-known that in excess it can cause teratogenesis in fish and birds. In this study we compared dietary toxicity of elemental selenium nanoparticles (SeNPs) with selenite and selenomethionine (Se-Met). Japanese medaka (Oryzias latipes) was used as a laboratory model to determine Se effects on adults and their offspring. Adult females were individually exposed using a dry diet fortified with 0, 10 or 20 µg/g of the three Se species for 7 days and then allowed to breed for 3 days. Fertilization rate and the proportion of malformed offspring were examined. The three Se diets led to significant increase in maternal tissue Se concentration in the order of Se-Met >>selenite > SeNP. However, in terms of proportion of malformed offspring, the effect of Se-Met = selenite > SeNP. The malformations included pericardial edema and craniofacial changes, which were typical for Se toxicity. The mismatch of maternal ovary Se concentration and proportion of malformed offspring suggested total Se concentration is a poor predictor of toxicity and teratogenesis. Comparing expression of four genes related to oxidative stress in maternal tissue also showed that there were significant differences in expression patterns between three Se diets in the order of selenite = SeNP > Se-Met. Our results showed that SeNPs cause similar toxicity as other Se species but require further study to elucidate the underlying mechanism., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Lamin is an intermediate protein underlying the nuclear envelope and it plays a key role in maintaining the integrity of the nucleus. A defect in the processing of its precursor by a metalloprotease, ZMPSTE24, results in the accumulation of farnesylated prelamin in the nucleus and causes various diseases, including Hutchinson-Gilford progeria syndrome (HGPS). However, the role of lamin processing is unclear in fish species. Here, we generated zmpste24-deficient medaka and evaluated their phenotype. Unlike humans and mice, homozygous mutants did not show growth defects or lifespan shortening, despite lamin precursor accumulation. Gonadosomatic indices, blood glucose levels, and regenerative capacity of fins were similar in 1-year-old mutants and their wild-type (WT) siblings. Histological examination showed that the muscles, subcutaneous fat tissues, and gonads were normal in the mutants at the age of 1 year. However, the mutants showed hypersensitivity to X-ray irradiation, although p53target genes, p21 and mdm2, were induced 6 h after irradiation. Immunostaining of primary cultured cells from caudal fins and visualization of nuclei using H2B-GFP fusion proteins revealed an abnormal nuclear shape in the mutants both in vitro and in vivo. The telomere lengths were significantly shorter in the mutants compared to WT. Taken together, these results suggest that zmpste24-deficient medaka phenocopied HGPS only partially and that abnormal nuclear morphology and lifespan shortening are two independent events in vertebrates., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Accumulation of perfluorobutanesulfonate (PFBS) is frequently detected in biota, raising concerns about its ecological safety. However, hazardous effects of PFBS remain largely unexplored, especially for endocrine disrupting potency. In the present study, the multigenerational endocrine disrupting potential of PFBS was investigated by exposing F0 marine medaka eggs to PFBS at different concentrations (0, 1.0, 2.9, and 9.5 μg/L) until sexual maturity. The F1 and F2 generations were reared without continued exposure. Thyroidal disturbances were examined in all three generations. PFBS exposure decreased the levels of 3,5,3'-triiodothyronine (T3) in F0 female blood; however, it increased T3 or thyroxine (T4) levels in F0 brains, in which hyperthyroidism suppressed the local transcription of 5'-deiodinase 2 ( Dio2). Obviously decreased T3 was transferred to F1 eggs, although the parental influences were reversed in F1 larvae. Delayed hatching was coupled with elevated T3 levels in F1 larvae. F1 adults showed comparable symptoms of thyroidal disruption with F0 adults. A slight recovery was noted in the F2 generation, although F2 larvae still exhibited thyroid disruption and synthesized excessive T4. Our results suggested that the offspring suffered more severe dysfunction of the thyroidal axis albeit without direct exposure. This study provided the first molecular insight about PFBS toxicology on the thyroid, beneficial to both human and environmental risk assessment.

Many anthropogenic pollutants in coastal marine environments can induce immune impairments in wild fish and reduce their survival fitness. There is a pressing need to establish sensitive and high throughput in vivo tools to systematically evaluate the immunosuppressive effects of contaminants in marine teleosts. This study reviewed a battery of in vivo immune function detection technologies established for different biological hierarchies at molecular (immune function pathways and genes by next generation sequencing (NGS)), cellular (leukocytes profiles by flow cytometry), tissues/organ system (whole adult histo-array), and organism (host resistance assays (HRAs)) levels, to assess the immune competence of marine medaka Oryzias melastigma. This approach enables a holistic assessment of fish immune competence under different chemical exposure or environmental scenarios. The data obtained will also be useful to unravel the underlying immunotoxic mechanisms. Intriguingly, NGS analysis of hepatic immune gene expression profiles (male > female) are in support of the bacterial HRA findings, in which infection-induced mortality was consistently higher in females than in males. As such, reproductive stages and gender-specific responses must be taken into consideration when assessing the risk of immunotoxicants in the aquatic environment. The distinct phenotypic sexual dimorphism and short generation time (3 months) of marine medaka offer additional advantages for sex-related immunotoxicological investigation.

Osteosarcoma (OS) is a highly aggressive pediatric cancer, characterized by frequent lung metastasis and pathologic bone destruction. Vascular endothelial growth factor A (VEGFA), highly expressed in OS, not only contributes to angiogenesis within the tumor microenvironment via paracrine stimulation of vascular endothelial cells, but also acts as an autocrine survival factor for tumor cell themselves, thus making it a promising therapeutic target for OS. CRISPR/Cas9 is a versatile genome editing technology and holds tremendous promise for cancer treatment. However, a major bottleneck to achieve the therapeutic potential of the CRISPR/Cas9 is the lack of in vivo tumor-targeted delivery systems. Here, we screened an OS cell-specific aptamer (LC09) and developed a LC09-functionalized PEG-PEI-Cholesterol (PPC) lipopolymer encapsulating CRISPR/Cas9 plasmids encoding VEGFA gRNA and Cas9. Our results demonstrated that LC09 facilitated selective distribution of CRISPR/Cas9 in both orthotopic OS and lung metastasis, leading to effective VEGFA genome editing in tumor, decreased VEGFA expression and secretion, inhibited orthotopic OS malignancy and lung metastasis, as well as reduced angiogenesis and bone lesion with no detectable toxicity. The delivery system simultaneously restrained autocrine and paracrine VEGFA signaling in tumor cells and could facilitate translating CRISPR-Cas9 into clinical cancer treatment., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Growing evidence suggests that the immune system of teleost is vulnerable to xenoestrogens, which are ubiquitous in the marine environment. This study detected and identified the major circulatory immune proteins deregulated by 17α-ethinylestradiol (EE2), which may be linked to fish susceptibility to pathogens in the marine medaka, Oryzias melastigma. Fish immune competence was determined using a host resistance assay to pathogenic bacteria Edwardsiella tarda. Females were consistently more susceptible to infection-induced mortality than males. Exposure to EE2 could narrow the sex gap of mortality by increasing infection-induced death in male fish. Proteomic analysis revealed that the major plasma immune proteins of adult fish were highly sexually dimorphic. EE2 induced pronounced sex-specific changes in the plasma proteome, with the male plasma composition clearly becoming "feminised". Male plasma was found to contain a higher level of fibrinogens, WAP63 and ependymin-2-like protein, which are involved in coagulation, inflammation and regeneration. For the first time, we demonstrated that expression of C1q subunit B (C1Q), an initiating factor of the classical complement pathway, was higher in males and was suppressed in both sexes in response to EE2 and bacterial challenge. Moreover, cleavage and post-translational modification of C3, the central component of the complement system, could be altered by EE2 treatment in males (C3dg down; C3g up). Multiple regression analysis indicated that C1Q is possibly an indicator of fish survival, which warrants further confirmation. The findings support the potential application of plasma immune proteins for prognosis/diagnosis of fish immune competence. Moreover, this study provides the first biochemical basis of the sex-differences in fish immunity and how these differences might be modified by xenoestrogens., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Environmentally induced alterations of the immune system during sensitive developmental stages may manifest as abnormalities in immune organ configuration and/or immune cell differentiation. These not only render the early life stages more vulnerable to pathogens, but may also affect the adult immune competence. Knowledge of these sensitive periods in fish would provide an important prognostic/diagnostic tool for aquatic risk assessment of immunotoxicants. The marine medaka Oryzias melastigma is an emerging seawater fish model for immunotoxicology. Here, the presence and onset of four potentially sensitive periods during the development of innate and adaptive cellular immune defence were revealed in O. melastigma: 1.) initiation of phagocyte differentiation, 2.) migration and expansion of lymphoid progenitor cells, 3.) colonization of immune organs through lymphocyte progenitors and 4.) establishment of immune competence in the thymus. By using an established bacterial resistance assay for O. melastigma, larval immune competence (from newly hatched 1dph to 14dph) was found concomitantly increased with advanced thymus development and the presence of mature T-lymphocytes. A comparison between the marine O. melastigma and the freshwater counterpart Oryzias latipes disclosed a disparity in the T-lymphocyte maturation pattern, resulting in differences in the length of T-lymphocyte maturation. The results shed light on a potential difference between seawater and freshwater medaka in their sensitivity to environmental immunotoxicants. Further, medaka immune system development was compared and contrasted to economically important fish. The present study has provided a strong scientific basis for advanced investigation of critical windows for immune system development in fish., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Nonmammalian vertebrates have the capacity of lifelong tooth replacement. In all vertebrates, tooth formation requires contact and interaction between the oral or pharyngeal epithelium and the underlying mesenchyme. To secure lifelong replacement, the presence of odontogenic stem cells has been postulated, particularly in the epithelial compartment. This study uses an advanced teleost fish species, the marine medaka Oryzias melastigma, a close relative to Oryzias latipes, to examine the expression and distribution of telomerase reverse transcriptase (Tert), the catalytic unit of telomerase, in developing pharyngeal teeth and to relate these data to the proliferative activity of the cells. The data are complemented by expression analysis of the pluripotency marker oct4 and bona fide stem cell marker lgr5. Tert distribution and tert expression in developing tooth germs show a dynamic spatiotemporal pattern. Tert is present first in the mesenchyme but is downregulated as the odontoblasts differentiate. In contrast, in the epithelial enamel organ, Tert is absent during early stages of tooth formation and upregulated first in ameloblasts. Later, Tert is expressed and immunolocalized throughout the entire inner enamel epithelium. The pattern of Tert distribution is largely mutually exclusive with that of proliferating cell nuclear antigen (PCNA) immunoreactivity: highly proliferative cells, as revealed by PCNA staining, are negative for Tert; conversely, PCNA-negative cells are Tert-positive. Only the early condensed mesenchyme is both Tert- and PCNA-positive. The absence of tert-positive cells in the epithelial compartment of early tooth germs is underscored by the absence of oct4- and lgr5-positive cells, suggesting ways other than stem cell involvement to secure continuous renewal.

Fish blood is one of the crucial tissues of innate immune system, but the full repertoire of fish serum components involved in antibacterial defense is not fully identified. In this study, we demonstrated that turbot serum, but not the heat-inactivated control, significantly reduced the number of Edwardsiella tarda (E. tarda). By conjugating serum proteins with fluorescent dyes, we showed that E. tarda were coated with multiple fish proteins. In order to identify these proteins, we used E. tarda to capture turbot serum proteins and subjected the samples to shotgun proteomic analysis. A total of 76 fish proteins were identified in high confidence, including known antimicrobial proteins such as immunoglobins and complement components. 34 proteins with no previously known immunological functions were also identified. The expression of one of these proteins, IQ motif containing H (IQCH), was exclusively in fish brain and gonads and was induced during bacterial infection. This approach also allowed the study of the corresponding proteomic changes in E. tarda exposed to turbot serum, which is a general decrease of bacterial protein expression except for an upregulation of membrane components after serum treatment. Interestingly, while most other known stresses stimulate bacterial antioxidant enzymes, fish serum induced a rapid suppression of antioxidant proteins and led to an accumulation of reactive oxygen species. Heat treatment of fish serum eliminated this effect, suggesting that heat labile factors in the fish serum overrode bacterial antioxidant defenses. Taken together, this work offers a comprehensive view of the interactions between fish serum proteins and bacteria, and reveals previously unknown factors and mechanisms in fish innate immunity., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

3,3'-Diindolylmethane (DIM) has been promoted as an effective chemopreventive and antifouling additive. However, the concurrent risks or side effects of DIM are not fully understood, especially on tissues responsive to estrogen. Therefore, this study employed marine medaka (Oryzias melastigma) as a test model to evaluate relative safety and explore mechanisms of toxic action of DIM on development and function of gonad after chronic (28days) aqueous exposure to relatively low doses (0μg/L or 8.5μg/L). Integration of comprehensive toxicogenomic analysis at the transcriptome and proteome levels with apical endpoints, such as production of eggs and swimming performance of larvae, elucidated the molecular linkage in gonad from bottom up along the reproductive adverse outcome pathway. A series of sequential changes at the transcript and protein levels were linked to lesser fecundity and viability of larvae exposed to DIM. Anomalous production of vitellogenin (VTG) and eggshell proteins in testis confirmed the estrogenic potency of DIM. In the ovary, although storage of VTG was greater, lesser expressions of cathepsin enzymes blocked cleavage and incorporation of VTG into oocytes as yolk, which acted together with lower eggshell proteins to inhibit maturation of primary oocyte and thus contributed to impairment of fecundity. Overall, this study demonstrated that exposure to DIM impaired reproductive fitness. Diverse molecular initiating changes in gonads were linked to apical endpoints that could be used in assessment of risks posed by DIM on gametogenesis. In combination with chemical stability and potent endocrine disruption, the results of this study can inform decisions about the use of DIM either as chemopreventive or antifouling agent., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Telomerase expression has long been linked to promotion of tumor growth and cell proliferation in mammals. Interestingly, telomerase activity (TA) has been detected in skeletal muscle for a variety of fish species. Despite this being a unique feature in fish, very few studies have investigated the potential role of TA in muscle. The present study was set to prove the concepts that muscle telomerase in fish is related to body growth, and more specifically, to muscle cell proliferation and apoptosis in vivo. Moreover, muscle TA can be influenced by biotic factors and modulated by environmental stress. Using three fish species, mangrove red snapper (Lutjanus argentimaculatus), orange-spotted grouper (Epinephelus coioides), and marine medaka (Oryzias melastigma), the present work reports for the first time that fish muscle TA was sensitive to the environmental stresses of starvation, foodborne exposure to benzo[a]pyrene, and hypoxia. In marine medaka, muscle TA was coupled with fish growth during early life stages. Upon sexual maturation, muscle TA was confounded by sex (female>male). Muscle TA was significantly correlated with telomerase reverse transcriptase (TERT) protein expression (Pearson correlation r=0.892; p≤0.05), which was coupled with proliferating cell nuclear antigen (PCNA) cell proliferation, but not associated with apoptosis (omBax/omBcl2 ratio) in muscle tissue. The results reported here have bridged the knowledge gap between the existence and function of telomerase in fish muscle. The underlying regulatory mechanisms of muscle TA in fish warrant further exploration for comparison with telomerase regulation in mammals., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Recent cross-generational studies in teleost fish have raised the awareness that high levels of benzo[a]pyrene (BaP) could affect skeletal integrity in the directly exposed F0 and their F1-F2. However, no further details were provided about the causes for abnormalities on the molecular and cellular level and the persistence of such sub-organismal impairments at the transgenerational scale (beyond F2). Adult Oryzias latipes were exposed to 1μg/L BaP for 21days. The F1-F3 were examined for skeletal deformities, histopathological alterations of vertebral bodies and differential expression of key genes of bone metabolism. Significant increase of dorsal-ventral vertebral compression was evident in ancestrally exposed larvae. Histopathological analysis revealed abnormal loss of notochord sheath, a lack of notochord epithelial integrity, reduced bone tissue and decreased osteoblast abundance. A significant downregulation of ATF4 and/or osterix and a high biological variability of COL10, coupled with a significant deregulation of SOX9a/b in the F1-F3 suggest that ancestral BaP exposure most likely perturbed chordoblasts, chondroblast and osteoblast differentiation, resulting in defective notochord sheath repair and rendering the vertebral column more vulnerable to compression. The present findings provide novel molecular and cellular insights into BaP-induced transgenerational bone impairment in the unexposed F3. From the ecological risk assessment perspective, BaP needs to be regarded as a transgenerational skeletal toxicant, which exerts a far-reaching impact on fish survival and fitness. Given that basic mechanisms of cartilage/bone formation are conserved between medaka and mammals, the results may also shed light on the potential transgenerational effect of BaP on the genesis of skeletal diseases in humans., (Copyright © 2015 Elsevier Inc. All rights reserved.)