Exposure effects of benzalkonium chloride (BAC) on gonadal physiology and fertility suppression in medaka (Oryzias latipes).

Benzalkonium chloride (BAC), a commonly used quaternary ammonium compound in various products like antiseptics, cosmetics, and disinfectants, has raised concerns due to its potential to contaminate aquatic environments and subsequently affect the reproductive performance of the organisms within those ecosystems. The article underscores a critical concern regarding the impact of BAC on aquatic ecosystems, particularly its effect on fish reproductive quality, using medaka (Oryzias latipes) as a model organism. Firstly, while measuring lethal dose of BAC in adult medaka, we observed a dose dependent mortality in BAC treated fish (100 and 200 ppm: 100%; 60 ppm: 51.7%; 30 ppm or less: no mortality at 24 h post treatment (hpt)) and calculated the LD50 at 96 hpt as 39.291 ppm (95% confidence interval: 28.817-53.570 ppm). Further, we assessed the molecular, cellular and histological changes through long-term exposure. Enlarged sperm pockets and reduced spermatocyte were seen in BAC exposed testis while no significant structural changes were observed in the ovaries. Following BAC exposure, drastic alterations in the gene expression and cellular localization related to sex, estrogen signaling, and autophagy were also noted from gonads and liver. Subsequently, using a short-term exposure analysis, we confirmed the sex and time responsive transcriptional kinetics and found that BAC sequentially affected the gonadal somatic cells followed by germ cell differentiation. Finally, using reproductively competent male and female medaka, we conducted progeny production and performance analysis and depicted a drastic reduction in fecundity, and fertilization and hatching rate, indicating adverse effects of BAC on reproductive success. Cumulatively, these findings emphasize the consequences of widespread use of BAC on reproductive security of aquatic animals and highlights the need for further research to comprehend the potential harm posed by such compounds to aquatic animal health and ecosystem integrity.

A Case of Fournier's Gangrene in the Course of Treatment of Advanced Hepatocellular Carcinoma with Molecular-targeted Therapy.

Lenvatinib is a molecular-targeted agent with proven efficacy against hepatocellular carcinoma (HCC). We herein report a case of lenvatinib-associated Fournier gangrene. A 66-year-old man with advanced hepatocellular carcinoma presented with a high fever 4 weeks after switching to lenvatinib. He had severe erythema in the inguinal region, and abdominal computed tomography revealed extensive emphysema and scrotal abscesses. He was diagnosed with Fournier's gangrene, and his symptoms were successfully treated with local debridement and antimicrobial therapy. Although reports of lenvatinib-associated Fournier's gangrene are rare, they should be kept in mind, as the condition could progress rapidly and have poor outcomes.

A Case of Secondary Growth Hormone Deficiency that Developed into Cirrhosis after Several Years of Interrupted Growth Hormone Replacement Therapy.

This case report describes a patient who received hormone replacement therapy for secondary panhypopituitarism and subsequently developed diabetes. His physician decided to discontinue growth hormone (GH) replacement, which was previously deemed contraindicated. Following the diagnosis of fatty liver, the patient began to exhibit liver damage that progressed over the ensuing years, ultimately leading to cirrhosis. Common factors linked to cirrhosis were excluded, leading to the belief that GH deficiency over several years was the primary contributor to cirrhosis. Therefore, when treating patients with GH insufficiency and diabetes, clinicians should carefully consider the potential implications of GH replacement therapy.

Modeling the SDF-1/CXCR4 protein using advanced artificial intelligence and antagonist screening for Japanese anchovy.

SDF-1/CXCR4 chemokine signaling are indispensable for cell migration, especially the Primordial Germ Cell (PGC) migration towards the gonadal ridge during early development. We earlier found that this signaling is largely conserved in the Japanese anchovy (Engraulis japonicus, EJ), and a mere treatment of CXCR4 antagonist, AMD3100, leads to germ cell depletion and thereafter gonad sterilization. However, the effect of AMD3100 was limited. So, in this research, we scouted for CXCR4 antagonist with higher potency by employing advanced artificial intelligence deep learning-based computer simulations. Three potential candidates, AMD3465, WZ811, and LY2510924, were selected and in vivo validation was conducted using Japanese anchovy embryos. We found that seven transmembrane motif of EJ CXCR4a and EJ CXCR4b were extremely similar with human homolog while the CXCR4 chemokine receptor N terminal (PF12109, essential for SDF-1 binding) was missing in EJ CXCR4b. 3D protein analysis and cavity search predicted the cavity in EJ CXCR4a to be five times larger (6,307 Å³) than that in EJ CXCR4b (1,241 Å³). Docking analysis demonstrated lower binding energy of AMD3100 and AMD3465 to EJ CXCR4a (Vina score -9.6) and EJ CXCR4b (Vina score -8.8), respectively. Furthermore, we observed significant PGC mismigration in microinjected AMD3465 treated groups at 10, 100 and 1 × 105 nM concentration in 48 h post fertilized embryos. The other three antagonists showed various degrees of PGC dispersion, but no significant effect compared to their solvent control at tested concentrations was observed. Cumulatively, our results suggests that AMD3645 might be a better candidate for abnormal PGC migration in Japanese anchovy and warrants further investigation.

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.

Evolutionary differentiation of androgen receptor is responsible for sexual characteristic development in a teleost fish.

Teleost fishes exhibit complex sexual characteristics in response to androgens, such as fin enlargement and courtship display. However, the molecular mechanisms underlying their evolutionary acquisition remain largely unknown. To address this question, we analyse medaka (Oryzias latipes) mutants deficient in teleost-specific androgen receptor ohnologs (ara and arb). We discovered that neither ar ohnolog was required for spermatogenesis, whilst they appear to be functionally redundant for the courtship display in males. However, both were required for reproductive success: ara for tooth enlargement and the reproductive behaviour eliciting female receptivity, arb for male-specific fin morphogenesis and sexual motivation. We further showed that differences between the two ar ohnologs in their transcription, cellular localisation of their encoded proteins, and their downstream genetic programmes could be responsible for the phenotypic diversity between the ara and arb mutants. These findings suggest that the ar ohnologs have diverged in two ways: first, through the loss of their roles in spermatogenesis and second, through gene duplication followed by functional differentiation that has likely resolved the pleiotropic roles derived from their ancestral gene. Thus, our results provide insights into how genome duplication impacts the massive diversification of sexual characteristics in the teleost lineage.

Development of a chub mackerel with less-aggressive fry stage by genome editing of arginine vasotocin receptor V1a2.

Genome editing is a technology that can remarkably accelerate crop and animal breeding via artificial induction of desired traits with high accuracy. This study aimed to develop a chub mackerel variety with reduced aggression using an experimental system that enables efficient egg collection and genome editing. Sexual maturation and control of spawning season and time were technologically facilitated by controlling the photoperiod and water temperature of the rearing tank. In addition, appropriate low-temperature treatment conditions for delaying cleavage, shape of the glass capillary, and injection site were examined in detail in order to develop an efficient and robust microinjection system for the study. An arginine vasotocin receptor V1a2 (V1a2) knockout (KO) strain of chub mackerel was developed in order to reduce the frequency of cannibalistic behavior at the fry stage. Video data analysis using bioimage informatics quantified the frequency of aggressive behavior, indicating a significant 46% reduction (P = 0.0229) in the frequency of cannibalistic behavior than in wild type. Furthermore, in the V1a2 KO strain, the frequency of collisions with the wall and oxygen consumption also decreased. Overall, the manageable and calm phenotype reported here can potentially contribute to the development of a stable and sustainable marine product.

Long-day stimulation increases thyroid-stimulating hormone expression and affects gonadal development in chub mackerel.

For seasonal breeders, photoperiodic changes are important signals that mark the start of the breeding season. Thyroid-stimulating hormone (TSH) is a glycoprotein hormone that not only promotes the secretion of thyroid hormone but also plays a key role in regulating seasonal reproduction in birds and mammals. However, whether TSH activation has been implicated as a seasonal indicator in fish breeding has not been fully investigated. In this study, we isolated tshb as a starting point to elucidate the effect of photoperiodic changes on the activation of the reproductive axis of chub mackerel. The isolated tshb was classified as tshba, which is widely conserved in vertebrates. The quantitative PCR results showed that tshb was strongly expressed in the pituitary. When female and male chub mackerel with immature gonads were reared for six weeks under different photoperiodic conditions, the gonads developed substantially in the long-day (LD) reared fish compared to those in the short-day reared fish. Real-time PCR results showed that the expression level of tshb in the pituitary gland was significantly elevated in the LD group. Although there was no difference in the gonadotropin-releasing hormone 1 gene expression level in the preoptic area of the brain, follicle-stimulating hormone and luteinizing hormone gene expression levels in the pituitary were also significantly elevated in the LD group. In conclusion, TSH is a potential mediator of seasonal information in the reproductive endocrine axis and may induce gonadal development during the breeding season of chub mackerel.

Sex Lethal Gene Manipulates Gonadal Development of Medaka, Oryzias latipes, through Estrogenic Interventions.

Germ cells are pivotal for gonadal sexuality maintenance and reproduction. Sex lethal (sxl), the somatic sex determining gene of Drosophila, is the known regulator and initiator of germ cell femininity in invertebrates. However, the role of the Sxl homologue has rarely been investigated in vertebrates. So, we used medaka to clarify the role of sxl in vertebrate gonadogenesis and sexuality and identified two Sxl homologues, i.e., Sxl1a and Sxl1b. We found that sxl1a specifically expresses in the primordial germ cells (PGC), ovary, (early gonia and oocytes), while sxl1b distributions are ubiquitous. An mRNA overexpression of sxl1a accelerated germ cell numbers in 10 DAH XY fish, and sxl1a knockdown (KD), on the other hand, induced PGC mis-migration, aberrant PGC structuring and ultimately caused significant germ cell reduction in XX fish. Using an in vitro promoter analysis and in vivo steroid treatment, we found a strong link between sxl1a and estrogenic germ cell-population maintenance. Further, using sxl1a-KD and erß2-knockout fish, we determined that sxl1 acts through erß2 and controls PGC sexuality. Cumulatively, our study highlights the novel role of sxl1a in germ cell maintenance and sexual identity assignment and thus might become a steppingstone to understanding the commonalities of animal sexual development.

CDK14 Promotes Axon Regeneration by Regulating the Noncanonical Wnt Signaling Pathway in a Kinase-Independent Manner.

The postinjury regenerative capacity of neurons is known to be mediated by a complex interaction of intrinsic regenerative pathways and external cues. In Caenorhabditis elegans, the initiation of axon regeneration is regulated by the nonmuscle myosin light chain-4 (MLC-4) phosphorylation signaling pathway. In this study, we have identified svh-16/cdk-14, a mammalian CDK14 homolog, as a positive regulator of axon regeneration in motor neurons. We then isolated the CDK-14-binding protein MIG-5/Disheveled (Dsh) and found that EGL-20/Wnt and the MIG-1/Frizzled receptor (Fz) are required for efficient axon regeneration. Further, we demonstrate that CDK-14 activates EPHX-1, the C. elegans homolog of the mammalian ephexin Rho-type GTPase guanine nucleotide exchange factor (GEF), in a kinase-independent manner. EPHX-1 functions as a GEF for the CDC-42 GTPase, inhibiting myosin phosphatase, which maintains MLC-4 phosphorylation. These results suggest that CDK14 activates the RhoGEF-CDC42-MLC phosphorylation axis in a noncanonical Wnt signaling pathway that promotes axon regeneration.SIGNIFICANCE STATEMENT Noncanonical Wnt signaling is mediated by Frizzled receptor (Fz), Disheveled (Dsh), Rho-type GTPase, and nonmuscle myosin light chain (MLC) phosphorylation. This study identified svh-16/cdk-14, which encodes a mammalian CDK14 homolog, as a regulator of axon regeneration in Caenorhabditis elegans motor neurons. We show that CDK-14 binds to MIG-5/Dsh, and that EGL-20/Wnt, MIG-1/Fz, and EPHX-1/RhoGEF are required for axon regeneration. The phosphorylation-mimetic MLC-4 suppressed axon regeneration defects in mig-1, cdk-14, and ephx-1 mutants. CDK-14 mediates kinase-independent activation of EPHX-1, which functions as a guanine nucleotide exchange factor for CDC-42 GTPase. Activated CDC-42 inactivates myosin phosphatase and thereby maintains MLC phosphorylation. Thus, the noncanonical Wnt signaling pathway controls axon regeneration via the CDK-14-EPHX-1-CDC-42-MLC phosphorylation axis.

The Integrin Signaling Network Promotes Axon Regeneration via the Src-Ephexin-RhoA GTPase Signaling Axis.

Axon regeneration is an evolutionarily conserved process essential for restoring the function of damaged neurons. In Caenorhabditis elegans hermaphrodites, initiation of axon regeneration is regulated by the RhoA GTPase-ROCK (Rho-associated coiled-coil kinase)-regulatory nonmuscle myosin light-chain phosphorylation signaling pathway. However, the upstream mechanism that activates the RhoA pathway remains unknown. Here, we show that axon injury activates TLN-1/talin via the cAMP-Epac (exchange protein directly activated by cAMP)-Rap GTPase cascade and that TLN-1 induces multiple downstream events, one of which is integrin inside-out activation, leading to the activation of the RhoA-ROCK signaling pathway. We found that the nonreceptor tyrosine kinase Src, a key mediator of integrin signaling, activates the Rho guanine nucleotide exchange factor EPHX-1/ephexin by phosphorylating the Tyr-568 residue in the autoinhibitory domain. Our results suggest that the C. elegans integrin signaling network regulates axon regeneration via the Src-RhoGEF-RhoA axis.SIGNIFICANCE STATEMENT The ability of axons to regenerate after injury is governed by cell-intrinsic regeneration pathways. We have previously demonstrated that the Caenorhabditis elegans RhoA GTPase-ROCK (Rho-associated coiled-coil kinase) pathway promotes axon regeneration by inducing MLC-4 phosphorylation. In this study, we found that axon injury activates TLN-1/talin through the cAMP-Epac (exchange protein directly activated by cAMP)-Rap GTPase cascade, leading to integrin inside-out activation, which promotes axonal regeneration by activating the RhoA signaling pathway. In this pathway, SRC-1/Src acts downstream of integrin activation and subsequently activates EPHX-1/ephexin RhoGEF by phosphorylating the Tyr-568 residue in the autoinhibitory domain. Our results suggest that the C. elegans integrin signaling network regulates axon regeneration via the Src-RhoGEF-RhoA axis.

Sex determination, gonadal sex differentiation, and plasticity in vertebrate species.

A diverse array of sex determination (SD) mechanisms, encompassing environmental to genetic, have been found to exist among vertebrates, covering a spectrum from fixed SD mechanisms (mammals) to functional sex change in fishes (sequential hermaphroditic fishes). A major landmark in vertebrate SD was the discovery of the SRY gene in 1990. Since that time, many attempts to clone an SRY ortholog from nonmammalian vertebrates remained unsuccessful, until 2002, when DMY/dmrt1by was discovered as the SD gene of a small fish, medaka. Surprisingly, however, DMY/dmrt1by was found in only 2 species among more than 20 species of medaka, suggesting a large diversity of SD genes among vertebrates. Considerable progress has been made over the last 3 decades, such that it is now possible to formulate reasonable paradigms of how SD and gonadal sex differentiation may work in some model vertebrate species. This review outlines our current understanding of vertebrate SD and gonadal sex differentiation, with a focus on the molecular and cellular mechanisms involved. An impressive number of genes and factors have been discovered that play important roles in testicular and ovarian differentiation. An antagonism between the male and female pathway genes exists in gonads during both sex differentiation and, surprisingly, even as adults, suggesting that, in addition to sex-changing fishes, gonochoristic vertebrates including mice maintain some degree of gonadal sexual plasticity into adulthood. Importantly, a review of various SD mechanisms among vertebrates suggests that this is the ideal biological event that can make us understand the evolutionary conundrums underlying speciation and species diversity.

Estrogen and estrogen receptors chauffeur the sex-biased autophagic action in liver.

Autophagy, or cellular self-digestion, is an essential cellular process imperative for energy homeostasis, development, differentiation, and survival. However, the intrinsic factors that bring about the sex-biased differences in liver autophagy are still unknown. In this work, we found that autophagic genes variably expresses in the steroidogenic tissues, mostly abundant in liver, and is influenced by the individual's sexuality. Starvation-induced autophagy in a time-dependent female-dominated manner, and upon starvation, a strong gender responsive circulating steroid-HK2 relation was observed, which highlighted the importance of estrogen in autophagy regulation. This was further confirmed by the enhanced or suppressed autophagy upon estrogen addition (male) or blockage (female), respectively. In addition, we found that estrogen proved to be the common denominator between stress management, glucose metabolism, and autophagic action in female fish. To understand further, we used estrogen receptor (ER)α- and ER-ß2-knockout (KO) medaka and found ER-specific differences in sex-biased autophagy. Interestingly, starvation resulted in significantly elevated mTOR transcription (compared with control) in male ERα-KO fish while HK2 and ULK activation was greatly decreased in both KO fish in a female oriented fashion. Later, ChIP analysis confirmed that, NRF2, an upstream regulator of mTOR, only binds to ERα, while both ERα and ERß2 are effectively pulled down the HK2 and LC3. FIHC data show that, in both ER-KO fish, LC3 nuclear-cytoplasmic transport and its associated pathways involving SIRT1 and DOR were greatly affected. Cumulatively, our data suggest that, ERα-KO strongly affected the early autophagic initiation and altered the LC3 nuclear-cytoplasmic translocation, thereby influencing the sex-biased final autophagosome formation in medaka. Thus, existence of steroid responsive autophagy regulatory-switches and sex-biased steroid/steroid receptor availability influences the gender-skewed autophagy. Expectedly, this study may furnish newer appreciation for gender-specific medicine research and therapeutics.

Leptin stimulates gonadotropin release and ovarian development in marine teleost chub mackerel.

Leptin transmits information about energy stored in the periphery to the reproductive axis and is an essential signal for puberty initiation in mammals; however, to date, few studies have focused on the direct effects of leptin stimulation on reproductive factors in fish. This study demonstrated the effect of leptin stimulation on important reproductive factors and ovarian development in the marine teleost chub mackerel (Scomber japonicus). We prepared recombinant leptin and conducted functional analyses through in vitro bioassays using primary pituitary cells, long-term leptin treatment administered to pre-pubertal females, and intracerebroventricular (ICV) administration. The results showed that leptin stimulation strongly induced gonadotropin (follicle-stimulating hormone: FSH and luteinizing hormone: LH) secretion from pituitary cells collected from pre-pubertal females, and that long-term leptin treatment significantly promoted ovarian development and triggered pubertal onset. Furthermore, ICV administration of leptin did not affect kisspeptin gene expression but significantly upregulated gonadotropin-releasing hormone 1 (gnrh1), fshb and lhb gene expression in sexually immature females. These results strongly suggest leptin as an important signal for reproductive-axis activation in chub mackerel.

Alanine scanning and characterization of core peptides in Scombridae fish family for construction of Kiss1 super analog.

Chronic Kiss1 administration strongly promotes gonadal development in immature chub mackerel (cm) (Scomber japonicus). Here, we performed an Alanine scanning (Ala-scanning) of Kiss1 to determine its key residues. Additionally, we examined functional peptides from 16 Scombridae species to develop maturation-inducing super-analogs that can be used universally in Scombridae species. In the Ala-scanning of Kiss1-15 (QDMSSYNFNSFGLRY), substitution of Gln1 and Asp2 did not affect agonistic activity. This suggests that peptides could be downsized. Furthermore, it is possible that Phe8 can be substituted by unnatural amino acids that are difficult to degrade. In molecular cloning, only Scomber showed a 16-residue form as a putative mature peptide. The other genera, did not have a His residue at the N-terminal, which indicated that the functional peptide was 15 residues and the second and third residues from the N-terminal showed variation between interspecies. Next, we examined the binding affinity of various synthetic Kiss1 core peptides in Scombridae interspecies using an SRE-Luc reporter system. We cloned Kiss1 receptors (KissR1) from bluefin tuna (bft) (Thunnus orientalis) and Japanese Spanish mackerel (jsm) (Scomberomorus niphonius) for the first time. In binding affinity with cmKissR1, bftKissR1, and jsmKissR1, the species specificity of the second residue from the N-terminus in each ligand could be ignored, but the difference in the third residue strongly affected receptor binding. Scombridae species possess the same Kiss1 system but the structure of the functional peptide might be species-specific.

Estrogen Receptor ß2 Oversees Germ Cell Maintenance and Gonadal Sex Differentiation in Medaka, Oryzias latipes.

In vertebrates, estrogen receptors are essential for estrogen-associated early gonadal sex development. Our previous studies revealed sexual dimorphic expression of estrogen receptor ß2 (ERß2) during embryogenesis of medaka, and here we investigated the functional importance of ERß2 in female gonad development and maintenance using a transgenerational ERß2-knockdown (ERß2-KD) line and ERß2-null mutants. We found that ERß2 reduction favored male-biased gene transcription, suppressed female-responsive gene expression, and affected SDF1a and CXCR4b co-assisted chemotactic primordial germ cell (PGC) migration. Co-overexpression of SDF1a and CXXR4b restored the ERß2-KD/KO associated PGC mismigration. Further analysis confirmed that curtailment of ERß2 increased intracellular Ca2+ concentration, disrupted intra- and extracellular calcium homeostasis, and instigated autophagic germ cell degradation and germ cell loss, which in some cases ultimately affected the XX female sexual development. This study is expected improve our understanding of germ cell maintenance and sex spectrum, and hence open new avenues for reproductive disorder management.

Occurrence of sul and tet(M) genes in bacterial community in Japanese marine aquaculture environment throughout the year: Profile comparison with Taiwanese and Finnish aquaculture waters.

The use of antibiotics in aquaculture causes selection pressure for antibiotic-resistant bacteria (ARB). Antibiotic resistance genes (ARGs) may persist in ARB and the environment for long time even after stopping drug administration. Here we show monthly differences in the occurrences of genes conferring resistance to sulfonamides (i.e. sul1, sul2, sul3), and tetracyclines (tet(M)) in Japanese aquaculture seawater accompanied by records of drug administration. sul2 was found to persist throughout the year, whereas the occurrences of sul1, sul3, and tet(M) changed month-to-month. sul3 and tet(M) were detected in natural bacterial assemblages in May and July, but not in colony-forming bacteria, thus suggesting that the sul3 was harbored by the non-culturable fraction of the bacterial community. Comparison of results from Taiwanese, Japanese, and Finnish aquaculture waters reveals that the profile of sul genes and tet(M) in Taiwan resembles that in Japan, but is distinct from that in Finland. To our knowledge, this work represents the first report to use the same method to compare the dynamics of sul genes and tet(M) in aquaculture seawater in different countries.

Molecular cloning, characterization and expression analysis of complement components in red sea bream (Pagrus major) after Edwardsiella tarda and red sea bream Iridovirus (RSIV) challenge.

The complement system plays an important role in immune regulation and acts as the first line of defense against any pathogenic attack. To comprehend the red sea bream (Pagrus major) immune response, three complement genes, namely, pmC1r, pmMASP and pmC3, belonging to the classical, lectin and alternative complement cascade, respectively, were identified and characterized. pmC1r, pmMASP, and pmC3 were comprised of 2535, 3352, and 5735 base mRNA which encodes 732, 1029 and 1677 aa putative proteins, respectively. Phylogenetically, all the three studied genes clustered with their corresponding homologous clade. Tissue distribution and cellular localization data demonstrated a very high prevalence of all the three genes in the liver. Both bacterial and viral infection resulted in significant transcriptional alterations in all three genes in the liver with respect to their vehicle control counterparts. Specifically, bacterial challenge affected the pmMASP and pmC3 expression, while the viral infection resulted in pmC1r and pmC3 mRNA activation. Altogether, our data demonstrate the ability of pmC1r, pmMASP and pmC3 in bringing about an immune response against any pathogenic encroachment, and thus activating, not only one, but all the three complement pathways, in red sea bream.

Identification of Quantitative Trait Loci for Resistance to RSIVD in Red Sea Bream (Pagrus major).

Red sea bream iridoviral disease (RSIVD) is a major viral disease in red sea bream farming in Japan. Previously, we identified one candidate male individual of red sea bream that was significantly associated with convalescent individuals after RSIVD. The purpose of this study is to identify the quantitative trait loci (QTL) linked to the RSIVD-resistant trait for future marker-assisted selection (MAS). Two test families were developed using the candidate male in 2014 (Fam-2014) and 2015 (Fam-2015). These test families were challenged with RSIV, and phenotypes were evaluated. Then, de novo genome sequences of red sea bream were obtained through next-generation sequencing, and microsatellite markers were searched and selected for linkage map construction. One immune-related gene, MHC class IIß, was also used for linkage map construction. Of the microsatellite markers searched, 148 and 197 were mapped on 23 and 27 linkage groups in the female and male linkage maps, respectively, covering approximately 65% of genomes in both sexes. One QTL linked to an RSIVD-resistant trait was found in linkage group 2 of the candidate male in Fam-2014, and the phenotypic variance of the QTL was 31.1%. The QTL was closely linked to MHC class IIß. Moreover, the QTL observed in Fam-2014 was also significantly linked to an RSIVD-resistant trait in the candidate male of Fam-2015. Our results suggest that the RSIVD-resistant trait in the candidate male was controlled by one major QTL closely linked to the MHC class IIß gene and could be useful for MAS of red sea bream.

Hatching enzymes disrupt aberrant gonadal degeneration by the autophagy/apoptosis cell fate decision.

Environmental stressors, gonadal degenerative diseases and tumour development can significantly alter the oocyte physiology, and species fertility and fitness. To expand the molecular understanding about oocyte degradation, we isolated several spliced variants of Japanese anchovy hatching enzymes (AcHEs; ovastacin homologue) 1 and 2, and analysed their potential in oocyte sustenance. Particularly, AcHE1b, an ovary-specific, steroid-regulated, methylation-dependent, stress-responsive isoform, was neofunctionalized to regulate autophagic oocyte degeneration. AcHE1a and 2 triggered apoptotic degeneration in vitellogenic and mature oocytes, respectively. Progesterone, starvation, and high temperature elevated the total degenerating oocyte population and AcHE1b transcription by hyper-demethylation. Overexpression, knockdown and intracellular zinc ion chelation study confirmed the functional significance of AcHE1b in autophagy induction, possibly to mitigate the stress effects in fish, via ion-homeostasis. Our finding chronicles the importance of AcHEs in stress-influenced apoptosis/autophagy cell fate decision and may prove significant in reproductive failure assessments, gonadal health maintenance and ovarian degenerative disease therapy.