Genetic architectures of postmating isolation and morphology of two highly diverged rockfishes (genus Sebastes).

Postmating isolation is thought to be an important driver of the late stages of speciation. However, relatively little is empirically known about the process compared with other isolating mechanisms that drive the early stages of speciation, especially in non-model organisms. We characterized the genetic architecture of postmating isolation between 2 rockfishes, Sebastes schlegelii and S. trivittatus, whose reproductive isolation is complete. We examined transmission ratio distortion (TRD) patterns of genetic markers in 2 reciprocal backcross populations. Markers showing either of the 2 types of TRD was widespread across the genome, with some of the distorted markers forming extensive clusters around the recombination coldspots. These suggest that the postmating isolation effectively prevents gene flow across the genome and the recombination landscape contributes to the genetic architecture. Comparisons between 2 backcross families and 2 developmental stages showed little similarity in the distorted markers, suggesting asymmetry and stage specificity of the isolation. This may be due to hybrid incompatibility involving maternal factors or extrinsic selection. The lack of sex-ratio distortion in the mapping families suggested that Haldane's rule in terms of hybrid inviability does not hold. Additionally, quantitative trait loci (QTL) mapping detected significant QTLs for sex and the morphological traits relevant to speciation and convergence of rockfishes, including body coloration. Genes in the melanocortin system, including agouti-signaling protein 1 (asip1) and melanocortin 1 receptor (mc1r), might underlie the horizontal and vertical color patterns on the body, respectively. These findings constitute an essential step toward a comprehensive understanding of speciation and morphological diversification of rockfishes.

Identification and characterization of lipocalin-type prostaglandin D2 synthase homologs in the urine of male rockfish.

Black rockfish (Sebastes schlegelii) and its relatives are viviparous marine fish. Males produce urinary proteins during the copulation season; however, the identity of these proteins was unknown. In this study, we focused on high-molecular-weight urinary proteins (HMWups) in male black rockfish. The HMWups were identified by liquid chromatography and tandem mass spectrometry (LC-MS/MS) of urine. In silico analyses of RNA-seq data predicted the tissue distribution of candidate HMWup transcripts and their gene structures. Candidate cDNAs were cloned and a recombinant protein of a major candidate was prepared. Western blotting of urine using an antiserum against the recombinant protein was performed to reconfirm the LC-MS/MS results. Quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry were employed to validate the prediction by RNA-seq and identify the cells producing HMWups, respectively. LC-MS/MS, in conjunction with Western blotting and cDNA cloning, identified the HMWups as lipocalin-type prostaglandin D2 synthase (l-PGDS) homologs. RNA-seq analyses and qRT-PCR revealed that the l-PGDS homolog transcripts were dominantly expressed in the testis and male kidney; Sertoli cells and epithelial cells in the renal tubules were immunoreactive. These results indicated that major protein components in the urine of male black rockfish are l-PGDS homologs, potentially produced by the renal tubules in the kidney. Male rockfish (genus Sebastes) are thought to release unknown pheromone substances during mating behavior. The knowledge and tools obtained in this study empower research into the role(s) of HMWups in pheromone systems underlying rockfish reproduction. No protein-type teleost pheromone has heretofore been discovered.

Changes in the Hepatic Expression of Three Vitellogenin Subtype Genes During Ovarian Development in Female White-Edged Rockfish (Sebastes taczanowskii).

Viviparous fish, including white-edged rockfish (Sebastes taczanowskii), accumulate substantial yolk mass in the oocytes; however, the details of the molecular mechanisms underlying yolk formation are not yet fully understood, especially concerning multiplicity in the yolk precursor vitellogenin (Vtg). The present study aimed to reveal the hepatic transcriptional profiles of multiple vtg gene transcripts (vtgAa, vtgAb, vtgC) during the reproductive cycle in captive female white-edged rockfish reared in an aquarium under natural photo-thermal conditions. The serum estradiol-17ß concentration and the hepatic transcript levels of all vtg subtypes increased with the progress of vitellogenesis; both levels decreased at the beginning of oocyte maturation and remained low during the gestation period. Considering the similarity in the transcriptional profiles of vtg subtypes between Sebastes and Oncorhynchus, along with the differences between Sebastes and Morone, it is suggested that the transcription patterns of multiple vtg genes relate to neither their reproductive modes (viviparity versus oviparity) nor to teleost phylogeny.

Molecular characterization of fshb and lhb subunits and their expression profiles in captive white-edged rockfish, Sebastes taczanowskii.

Fundamental knowledge on the regulation of reproduction by gonadotropins (Gths) is quite limited in viviparous fishes. In the present study, we performed molecular cloning and characterization of cDNAs encoding two Gth subunits (fshb and lhb) from the pituitaries of viviparous white-edged rockfish, Sebastes taczanowskii; expression profiles of both gene transcripts were elucidated in the pituitaries of reproductive males and females which were kept in a captive environment. The cloned fshb and lhb fragments exhibited high sequence identities with corresponding ß-subunit sequences from black rockfish, S. schlegelii. Notably, the fshb of white-edged rockfish appeared to lack a putative N-glycosylation site, whereas lhb conserved it. Expression of fshb and lhb transcripts in the rockfish pituitaries largely changed in synchrony but for minor exceptions. In males, levels of both transcripts increased with progression of spermatogenesis, although the peak for fshb (October) appeared slightly earlier than that for lhb (November). In females, both gene transcripts exhibited synchronous bimodal changes. High expression of fshb and lhb transcripts in the female pituitary during the gestation period, followed by the drastic decrease at parturition, suggest their possible involvement in regulation of gestation of this species. The knowledge gained for Sebastes in this study superimposes fundamental information necessary for further physiological understanding of viviparity in teleost fish.

Complex film of chitosan and carboxymethyl cellulose nanofibers.

A polymer film composed of a mixture of chitosan (Ch) and carboxymethyl cellulose sodium salt (CMC) nanofibers was deposited on a glass surface. The thin film of the Ch-CMC mixture obtained was stable, and fibroblast adhesion to the film was lowest when the weight ratio of Ch to CMC was 4:6. The ζ-potential and contact angle of the mixture film indicated that a polyion complex of Ch and CMC was formed. The mechanical strength of the film composed of Ch-CMC nanofiber complexes was much higher than that of the film composed of Ch-water-soluble CMC complexes (non-nanofiber), likely because the entanglement of nanofibers was enhanced by electrostatic attractions. These results indicate that the charge-neutralized nanofiber film was highly effective in suppressing cell adhesion and therefore is a promising material for biomedical applications.