dnd is a germline-specific maternal RNA expressed in various vertebrate classes, which encodes an RNA-binding protein that is essential for PGC migration. The purpose of this study is fundamental research about starry flounder dnd gene for germ cell marker development. In this study, we cloned and analyzed the expression levels of Platichthys stellatus dead end (psdnd) in various tissues and embryonic stages. The psdnd gene was isolated from starry flounder ovaries, cloned into a pGEM-t vector, and sequenced. Full-length of psdnd cDNA was 1495 bp long, encoding 395 amino acids. psdnd expression levels were investigated by real-time polymerase chain reaction (qRT-PCR) in various tissues and embryo developmental stages. psdnd transcripts were detected in the testes and ovaries, but not in somatic tissues. Embryonic psdnd expression levels were higher during early embryo development stages than during late embryogenesis; psdnd expression was highest at the 1 cell stage, then gradually decreased throughout the subsequent developmental stages. The spatial expression pattern was analyzed by whole-mount in situ hybridization (WISH). The psdnd transcripts migration pattern was similar with zebrafish (Danio rerio). Our results suggest that psdnd may function as a germ cell-specific marker.

The roughscale sole, Clidoderma asperrimum is categorized as an endangered species. Sperm freezing is essential for preserving gametes. This study examined the CPA concentration, diluent, dilution ratio, and thawing temperature to design a sperm cryopreservation protocol for roughscale sole. The variables examined included sperm motility and kinematics, cell survival, fertilization, and DNA fragmentation. Sperm motility parameters were assessed via computer-assisted sperm analysis using a CEROS II instrument. Cell survival rate and DNA damage were assessed using the Cell Counting Kit-8 and single-cell gel electrophoresis assay, respectively. Sperm preservation was tested using several CPAs, including ethylene glycol, dimethyl sulfoxide (DMSO), glycerol, propylene glycol, and methanol. The diluents tested were 300 mM sucrose, 300 mM glucose, Stein’s solution, Ringer’s solution, and Hank’s solution. The optimal conditions for sperm cryopreservation were 10% DMSO + Stein’s solution. After thawing, sperm motility was highest with a 1:1 dilution ratio (sperm to CPA + diluent), at 69.20 ± 0.32%; thawing at 10 °C was optimal for post-thaw motility (72.03 ± 0.95%). The highest fertilization rate (40.00 ± 1.22%) was obtained using DMSO. The fresh sperm had the lowest tail DNA, followed by 10% DMSO + Stein’s solution. The developed cryopreservation methods can be used in roughscale sole hatcheries.

A diffuser-incorporated transmission near-infrared (NIR) scheme that enables direct spectral collection of packed granular samples with reliable sample representation and reproducibility has been demonstrated. The analytical utility of this method has been evaluated for the determination of polyethylene (PE) pellet density and the discrimination of the geographical origin of rice samples. Based on the preliminary observation of transmission spectral features acquired from spherical polyoxymethylene (POM) packings composed of different particle sizes as well as packing thickness, a portion of the radiation was propagated through the void space in the packing without fully interacting with the POM pellets. This type of radiation, so-called non-fully interacted radiation (NFIR), adversely affected the sample representation as well as the reproducibility of transmission measurements. To maximize the interaction of NIR radiation with granular samples, a polytetrafluoroethylene (PTFE) diffuser was positioned in front of the sample packing to introduce isotropically diffused radiation into the sample. This diffuser-incorporated scheme resulted in highly reproducible transmission spectra for both packed granular samples. Consequently, the density determination of PE pellets as well as discrimination of rice samples according to geographical origin was more accurate using the proposed scheme.

Intermediate-term preservation of sperm assists the reproductive management of fish spermatozoa; however, no information is available on sperm of the spotted halibut, Verasper variegatus. We aimed to identify the optimum diluents, temperatures, dilution ratios, antibiotics, and antioxidants for sperm motility and cell viability. The diluents evaluated were marine fish Ringer’s solution (MFRS), Stein’s solution, 300 mM sucrose, and 300 mM glucose (diluted 1:1 [sperm: diluent], 1:2, 1:4, and 1:10 and stored at 0, 2, 4, and 6 °C). Neomycin and gentamycin (100, 200, 400, and 800 mg/L) and antioxidants (Mito-TEMPO [0, 25, 50, 75, 100, 125, 150, 175, and 200 µM], reduced glutathione [0, 2, 4, 6, 8, and 10 mM], and trehalose [0, 50, 100, 150, 200, and 250 mM]) were assessed in terms of sperm preservation. The most effective condition for cold storage of spotted halibut sperm was Stein’s solution at a dilution ratio of 1:4 at 2 °C, with a combination of neomycin 800 mg/L and 250 mM trehalose that showed spermatozoa motility of > 43% after 60 days. These storage conditions will be valuable for spotted halibut hatcheries.

The starry flounder Platichthys stellatus, an economically important benthic fish species widely distributed throughout the North Pacific Ocean, exhibits poor heat tolerance, resulting in frequent adult mass mortality in aquaculture settings. Heat stress disrupts the energy metabolism of P. stellatus, elevating blood glucose and implying its sensitivity to rising water temperatures. In particular, the survival of adult fish in aquaculture settings during the summer season is less than that of larvae or juveniles. This study aimed to understand the heat stress‐associated survival of adults by focusing on the molecular pathways of glycolysis/gluconeogenesis using comparative transcriptome analysis. Transcriptomes were obtained by RNA sequencing of P. stellatus livers of adults exposed to 16 and 28°C, together with juveniles at the same conditions. A total of 16,121,503–18,774,222 raw paired‐end reads were obtained after trimming. The assembled unigenes (39,489 out of 127,460) were annotated in the nr, Clusters of Orthologous Groups (COG), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Swiss‐Prot databases. A total of 6,858 differentially expressed genes were found in juveniles and adults at control (16°C) and high (28°C) water temperatures. Juveniles and adults had 1,856 genes downregulated and 1,548 genes upregulated at the 16°C control water temperature and 1,055 genes downregulated and 1,231 genes upregulated at 28°C, overall involving at least 47 KEGG pathways. When raising the water temperature and comparing the DEGs of juveniles and adults, respectively, we found that both groups showed similar upregulation patterns for the major heat shock protein (HSP) genes under temperature‐increasing conditions, indicating a conserved response mechanism. Additionally, both juvenile and adult fish showed similar upregulation of ER stress‐related genes, including endoplasmic reticulum chaperone BiP (BiP) and homocysteine‐responsive endoplasmic reticulum‐resident ubiquitin‐like domain member 1 (HERP), suggesting consistent transcriptional changes associated with ER stress under heat stress conditions. However, notable differences were observed in the expression of inflammation and immune response‐related genes, and juvenile P. stellatus showed upregulation of immune costimulatory protein B7‐H3 (B7H3) and immunoglobulin superfamily member 9 (IGSF9), while adults showed upregulation of high‐affinity immunoglobulin epsilon receptor subunit gamma (FCER1G), V‐type immunoglobulin domain‐containing suppressor of T‐cell activation (VSIR), and immunoglobulin superfamily DCC subclass member 3 (IGDCC3). Especially, phosphoglucomutase (PGM), glucose‐6‐phosphatase catalytic subunit (G6PC), phosphofructokinase (PFK), and bisphosphoglycerate mutase (BPGM), the enzymes involved in the glycolysis/gluconeogenesis pathway, showed significantly higher FPKM in adult P. stellatus than in juveniles. Overall, this study provides valuable insights into the transcriptional responses of P. stellatus to heat stress, which differ with age and may inform the development of strategies to improve the survival and productivity of this economically important fish species in aquaculture.