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The seed production of small yellow croaker (SYC) is constrained by reproductive dysfunction in captive-reared females. Reproductive dysfunction is closely linked to endocrine reproductive mechanisms. To better understand the reproductive dysfunction in captive broodstock, functional characterization of gonadotropins (GtHs: follicle stimulating hormone β subunit, fshβ; luteinizing hormone β subunit, lhβ; and glycoprotein α subunit, gpα) and sex steroids (17β-estradiol, E2; testosterone, T; progesterone; P) was performed using qRT-PCR, ELISA, in vivo, and in-vitro assay. The pituitary GtHs and gonadal steroids levels were significantly higher in ripen fish of both sexes. However, changes in lhβ and E2 levels in females were not significant in the developing and ripen stages. Furthermore, GtHs and steroids levels were lower in females compared to males throughout the reproductive cycle. In vivo administration of gonadotropin releasing hormone analogue (GnRHa) significantly increased the expression of GtHs in both dose- and time-related manners. The lower and higher doses of GnRHa led to successful spawning in male and female SYC, respectively. Sex steroids in vitro significantly inhibited the expression of lhβ in female SYC. Overall, GtHs were shown to play a vital role in final gonadal maturation, while steroids promoted negative feedback in the regulation of pituitary GtHs. Lower levels of GtHs and steroids might be key components in the reproductive dysfunction of captive-reared female SYC.

Female cleaner shrimps Lysmata amboinensis either underwent eyestalk ablations (EA), or were treated with two different wavelength intensities of red light (peak at 630 nm), and then their sex maturation and the effects on their egg quality parameters were compared. Vitellogenesis-inhibiting hormone (VIH), vitellogenin (VTG), VTG-receptor (VTG-R), and crustacean hyperglycemic hormone (CHH) expressions in the hepatopancreas were analysed. VTG-R expression (in situ) levels that caused the EA and red irradiation to induce the synthesis of VTG-R were assessed, and the triglycerides (TG) and glucose levels in the eggs were analysed. VIH expression was decreased in all experimental groups, but VTG and VTG-R expressions were increased. CHH expression and TG and glucose concentrations were higher in the red irradiation group, compared to the EA group. These results confirmed that the red light not only had a positive effect on the promotion of sex maturation, but it also improved the quality of the eggs, when compared to the EA method.

This study aimed to improve a sperm cryopreservation protocol for farmed Pacific abalone, Haliotis discus hannai. Dimethyl sulfoxide (Me2SO), glycerol, ethylene glycol (EG), propylene glycol (PG), and methanol were chosen as cryoprotectants (CPAs). Four different equilibration time (5, 10, 30, and 60 min), and two types of equilibration temperature (4 °C and 20 °C) were selected at the present experiment. Most equilibration temperatures with each CPA showed significant differences among different equilibration time. Post-thaw sperm motility of five CPAs showed no significant difference at two equilibration temperature. Based on these results, 8% Me2SO, 8% EG, 6% PG, 2% glycerol, and 2% methanol were chosen to determine optimal conditions for sperm cryopreservation of H. discus hannai. The highest post-thaw sperm motility (8% Me2SO: 50.6%, 8% EG: 45.6%, 2% glycerol: 44.5%, 6% PG: 28.7%, 2% methanol: 25.4%) was achieved after exposing sperm to liquid nitrogen (LN2) vapor for 10 min at 5 cm above the LN2 surface and then submerging them in LN2 for at least 2 h followed by thawing at 60 °C with seawater and recovering them at 20 °C with seawater. In this study, 8% Me2SO and 2% glycerol were chosen to check post-thaw sperm quality to estimate percentages of plasma membrane integrity (PMI), mitochondrial potential analysis (MP), and acrosome integrity (AI) using fluorescent techniques. No significant difference in PMI, MP, and AI was found between sperm cryopreserved with 8% Me2SO and those cryopreserved with 2% glycerol. The current study has demonstrated that 8% Me2SO was optimal for sperm cryopreservation for H. discus hannai with 5 min of equilibration time, 5 cm of rack height and 60 °C of thawing temperature. The present research provides more effective cryopreservation methods for H. discus hannai sperm than previous studies.

Bay scallops were exposed to four BaP concentrations (0.5, 1.0, 10 and 50 μg/L) for 72 h to elucidate their immune response. Immune parameters were evaluated by measuring nitric oxide (NO) levels in hemolymph. Additionally, we measured peptidoglycan recognition proteins (PGRP), fibrinogen-domain-containing protein (FReDC1), metallothionein (MT), and heat shock protein (HSP) 70 mRNA expression in digestive diverticula. NO as well as FReDC1 and MT expression in each BaP group increased significantly over time except for the BaP 0.5 group. The PGRP and HSP70 mRNA expression in the BaP 50 group increased in the range 6-24 h and then decreased. In situ hybridization also confirmed that there was higher MT mRNA expression in the BaP 50 group than in the control group at 72 h. Our results suggest that higher levels of BaP dampened scallop immune responses, while simultaneously reducing their ability to cope with oxidative stress and DNA damage. BaP exposure can be considered a potential immune inducer in bay scallop.

Tropomyosin (TPM) is a contractile protein responsible for muscle contraction through its actin-binding activity. The complete sequence of TPM in Haliotis discus hannai (Hdh-TPM) was 2160 bp, encoding 284 amino acids, and contained a TPM signature motif and a TPM domain. Gene ontology (GO) analysis based on the amino acid sequence predicted Hdh-TPM to have an actin-binding function in the cytoskeleton. The 3D analysis predicted the Hdh-TPM to have a coiled-coil α-helical structure. Phylogenetically, Hdh-TPM formed a cluster with other TPM/TPM1 proteins during analysis. The tissue-specific mRNA expression analysis found the higher expression of Hdh-TPM in the heart and muscles; however, during embryonic and larval development (ELD), the higher expression was found in the trochophore larvae and veliger larvae. Hdh-TPM expression was upregulated in fast-growing abalone. Increasing thermal stress over a long period decreased Hdh-TPM expression. Long-term starvation (>1 week) reduced the mRNA expression of Hdh-TPM in muscle; however, the mRNA expression of Hdh-TPM was significantly higher in the mantle, which may indicate overexpression. This study is the first comprehensive study to characterize the Hdh-TPM gene in Pacific abalone and to report the expression of Hdh-TPM in different organs, and during ELD, different growth patterns, thermal stress, seasonal changes, and starvation.

Neuropeptides play a central role in the regulation of reproduction, growth, development, and various other physiological processes. In our study, 26 neuropeptide precursors were identified from the pleuropedal and cerebral ganglia of Pacific abalone using transcriptome analysis. Of them, two (neuromacin and neuroglian) were potentially novel and two (myomodulin and FMRFamide) were involved in the reproductive regulation of mollusks. A BLAST search indicated that most of these neuropeptides exhibited the highest homologies with neuropeptides of other protostomian species. Myomodulin, FMRFamide, and molluscan insulin-related peptides shared 87%, 72%, and 88% sequence identities with homologous peptides of the tropical gastropod mollusk, Haliotis asinina. In silico analysis revealed that these identified neuropeptide precursors were likely to be extracellular secreted proteins. Alignment of multiple cerebrin, FFamide, and insulin-related peptide sequences illustrated that most of their biologically active residues were highly conserved with other invertebrate homologous neuropeptide residues. Three-dimensional (3D) structures of adipokinetic hormone, allatostain (A, B, and C), allatotropin, cerebrin, clionin, conopressin, elevenin, and neuromycin precursors in H. discus hannai exhibited a helix-loop-helix structure. Based on phylogenetic analysis, buccalin A1 and A2, allatostain B, and FCAP neuropeptide precursors of Pacific abalone formed a clade with other gastropod and bivalve mollusks neuropeptide precursors. This study provides a novel insight for further functional studies of abalone and other gastropod mollusks.

In this present study, we examined the anti-inflammatory and anti-oxidative properties of alcoholic lemon myrtle extract (LME). The total polyphenol and flavonoid content of LME were determined as 118.77 and 14.53 mg/g extract, respectively. LME showed anti-oxidative properties, such as 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) and 2,2′-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS) radical scavenging activity. The anti-inflammatory activities of LME were investigated using the lipopolysaccharide-stimulated murine macrophage RAW 264.7 cells. Pretreatment with LME was performed at non-cytotoxic concentrations of 10–100 μg/mL. LME inhibited the production of inflammatory mediators such as nitric oxide (NO). Enzyme-linked immunosorbent assay and reverse-transcriptase polymerase chain reaction (RT-PCR) revealed that pretreatment with LME suppressed the protein expression and mRNA levels of pro-inflammatory cytokines such as interleukin IL-6, and tumor necrosis factor (TNF)-α in a concentration-dependent manner, respectively. These results suggest that LME could be used as a potential therapeutic agent having potent anti-inflammatory effects that could be used to treat inflammatory bowel disease.

Serotonin receptor (5-HT) is a biogenic amine acting as a neurotransmitter and neuromodulator that mediates various aspects of reproduction and gametogenesis. The full-length nucleotide sequence of Haliotis discus hannai encodes a protein of 417 amino acids with a predicted molecular mass of 46.54 kDa and isoelectric point of 8.94. The structural profile of 5-HTHdh displayed key features of G protein-coupled receptors, including seven hydrophobic transmembrane domains, putative N-linked glycosylation sites, and several phosphorylation consensus motifs. It shares the highest homology of its amino acid sequence with the 5-HT receptor from Haliotis asinina, and to lesser extent of human 5-HT receptor. The cloned sequence possesses two cysteine residues (Cys-115 and Cys-193), which are likely to form a disulfide bond. Phylogenetic comparison with other known 5-HT receptor genes revealed that the 5-HTHdh is most closely related to the 5-HTHa receptor. The three-dimensional structure of the 5-HTHdh showed multiple alpha helices which is separated by a helix-loop-helix (HLH) structure. Quantitative PCR demonstrated that the receptor mRNA was predominantly expressed in the pleuropedal ganglion. Significant differences in the transcriptional activity of the 5-HTHdh gene were observed in the ovary at the ripening stage. An exclusive expression was detected in pleuropedal ganglion, testis, and ovary at higher effective accumulative temperature (1000 °C). In situ hybridization showed that the 5-HTHdh expressing neurosecretory cells were distributed in the cortex of the pleuropedal ganglion. Our results suggest that 5-HTHdh synthesized in the neural ganglia may be involved in oocyte maturation and spawning of H. discus hannai.

A carbonic anhydrase VII gene, encoding 277 amino acids, was identified in the intestinal tissue of pufferfish (Takifugu rubripes). The translated protein with an 833-bp complete coding sequence derived from the 1378-bp cloned sequence showed 83% identity with swamp eel CA VII, 76% with zebrafish CA VII, and 77% with coho salmon CA VII-like protein. The cloned protein also showed 68–69% identity with mammalian CA VII. The predicted molecular weight and iso-electric point of the protein were 30.84 kDa and 6.07, respectively. Active site analysis of the pufferfish CA VII indicated that most of the important residues involved in catalytic activity were highly conserved, whereas four cysteine residues at positions 55, 103, 184, and 275 differed from those in human CA II, and were related to cell-defense mechanisms against oxidative damage. Phylogenetic analysis showed that the cloned sequence was clustered within the fish CA VII clade and close to the swamp eel CA VII. Structural modeling of the pufferfish CA VII protein revealed the conservation of zinc binding histidine residues (zinc ion and histidine residues). Differential expression patterns of the pufferfish CA VII were determined with semiquantitative reverse transcription (RT)-PCR and quantitative PCR (q-PCR) as well. The results of q-PCR revealed that the pufferfish CA VII was highly expressed in intestinal tissue. The pufferfish CA VII was also detected within intestinal tissue sections using an in situ hybridization assay.

Pacific abalone (Haliotis discus hannai) is a commercially important high valued molluscan species. Its wild population has decreased in recent years. It is widely cultured in Korea. Traditional breeding programs have been implemented for hatchery production of abalone seeds. To obtain more genetic information for its molecular breeding program, a high-density linkage map and quantitative trait locus (QTL) for three growth-related traits was constructed for Pacific abalone. F1 cross population with two parents were sampled to construct the linkage map using genotyping by sequencing (GBS). A total of 664,630,534 clean reads and 56,686 SNPs were generated and 3,345 segregating SNPs were used to construct a consensus linkage map. The map spanned 1,747.023 cM with 18 linkage groups and an average interval of 0.55 cM. QTL analysis revealed two significant QTL in LG10 on the consensus linkage map of each growth-related trait. Both QTLs were located in the telomere region of the chromosome. Moreover, four potential candidate genes for growth-related traits were identified in the QTL region. Expression analysis revealed that these identified genes are involved in growth regulation of abalone. The newly constructed genetic linkage map, growth-related QTLs and potential candidate genes identified in the present study can be used as valuable genetic resources for marker-assisted selection (MAS) of Pacific abalone in molecular breeding program.

Temperature has crucial effects on gonadal development and reproduction of abalone. To understand the impact of thermal stress on molecular and physiological processes triggering the regulation of reproduction, changes in the mRNA expression of neuroendocrine genes encoding two abalone gonadotropin-releasing hormone (Hdh-GnRH, Hdh-GnRH-like peptide), GnRH receptor (Hdh-GnRH-R), Hdh-APGWamide, serotonin receptor (5-HThdh), and a heat shock protein HSP70 were examined in ganglia and testis of male Pacific abalone (Haliotis discus hannai). Abalone were exposed to low water temperature (LWT) and high water temperature (HWT) in early and peak breeding seasons for 7 days. Then, gonadosomatic index (GSI) was calculated, relative gene expression was measured by qRT-PCR, and levels of testosterone in hemolymph were also measured by ELISA during the peak breeding season. GSI did not show any significant changes during the early breeding season. However, it was significantly decreased in LWT- or HWT-exposed abalone compared to the normal water temperature (NWT) group during the peak breeding season. In the early breeding season, changes of mRNA expression of all five genes were significant between LWT and HWT groups on day-7. In the peak breeding season, compared to the NWT group, the mRNA expressions of different genes were significantly decreased in different tissues both in LWT and HWT groups of abalone, such as Hdh-GnRH-like peptide in the cerebral ganglion (CG) and testis; Hdh-GnRH in the pleuropedal ganglion (PPG) and branchial ganglion (BG); Hdh-GnRH-R in the CG, PPG, and testis; and Hdh-APGWamide in the PPG and testis. Interestingly, the expression of 5-HThdh was significantly increased in the HWT group but decreased in the LWT group. Expression of HSP70 was significantly increased in the testis after exposure to HWT. Hemolymph levels of testosterone were significantly decreased in the HWT group compared to those in the NWT group. Altogether, these results denote that thermal stress has a repressive effect on gonadal maturation and reproduction by regulating the expression of Hdh-GnRH-like peptide, Hdh-GnRH, Hdh-GnRH-R, Hdh-APGWamide, 5-HThdh, and HSP70 genes and levels of hemolymph testosterone.

Reproductive hormones play essential roles in the control of reproduction and gonadal maturation in fish. The purpose of this study was to determine the effects of cortisol administration (10 µg/g or 50 µg/g) or red light irradiation at two intensities (0.5 W/m2 or 1.0 W/m2) on the reproductive hormones in goldfish (Carassius auratus). The effects of different treatments were analyzed by determining the mRNA expression levels of gonadotropin-inhibitory hormone receptor (GnIH-R), chicken gonadotropin-releasing hormone (cGnRH-II), salmon GnRH (sGnRH), FSHβ, LHβ, and plasma testosterone and the level of 17β-estradiol for 48 h. Additionally, by double immunofluorescence staining, we detected the expression of both GnIH and GnRH in the diencephalons of goldfish brains. The mRNA expression of GnIH-R was significantly higher in the cortisol group and red light-irradiated group from 3 to 48 h than in the control group. Additionally, the mRNA levels of cGnRH-II, sGnRH, FSHβ, LHβ, testosterone, and 17β-estradiol were significantly lower in the cortisol group than in the other groups from 3 to 48 h. These results indicated that both cortisol and red light-emitting diode (LED) light increased GnIH expression and inhibited GnRH expression. In particular, red light irradiation suppressed reproductive responses as much as the cortisol treatment at 48 h. Thus, it could be an alternative method for suppressing reproductive responses in future aquacultures.

Water temperature is a crucial environmental factor that influences reproductive function of abalone. Broodstock conditioning exposed to effective accumulative temperature (EAT) is a common practice in abalone hatcheries. To understand the molecular mechanism underlying the regulation of gonadal maturation and reproduction of Haliotis discus hannai exposed to EAT and induced spawning period, changes in expression of neuroendocrine genes encoding two gonadotropin releasing hormone (Hdh-GnRH, GnRH-like peptide), GnRH receptor (HdhGnRH-R), serotonin receptor (5-HTHdh) and Hdh-APGWamide in neural ganglia and gonadal tissues were examined. Gonadosomatic index (GSI) was significantly increased with increasing EAT °C-days. Expression levels of Hdh-GnRH, GnRH-like peptide, HdhGnRH-R, 5-HTHdh and Hdh-APGWamide mRNA were significantly increased with increasing EAT °C-days in ganglion (where the gene synthesized) and gonadal tissues. The significant increase in mRNA expression of each examined gene started from EAT 500 to 750°C-days, reached an initial peak at 1000°C-days, suggesting gonadal maturation started from the onset of EAT and slowly continued until 750°C-days, then at 1000°C-days reached to initial peak developmental period. The maturation reached to spawning state at 1000°C-days and peaked at 1500°C-days. Hdh-GnRH showed significantly higher mRNA expression in pleuropedal ganglion and branchial ganglion, whereas GnRH like peptide showed higher expression in cerebral ganglion, and HdhGnRH-R, 5-HTHdh and Hdh-APGWamide showed higher expression in pleuropedal ganglion. All genes were expressed higher at higher EAT °C-days. During induced spawning period, higher mRNA expression of examined genes was observed at the time of spawning; however, a sharp decrease occurred after spawning, suggesting that these genes are involved in spawning activities. Taken together, these results indicate that an increase of EAT °C-days can increase expression of neuroendocrine genes and enhance gonadal maturation. Besides all these genes are involved in the process of spawning induction, and increase of GSI has a positive correlation with the increase of gene expression.

Insulin-like growth factor binding proteins (IGFBPs) are secreted proteins that play an important role in IGF regulation of growth and development of vertebrate and invertebrates. In this study, the IGFBP7 gene was cloned and characterized from mantle tissues of H. discus hannai, and designated as Hdh IGFBP7. The full-length cDNA sequence transcribed from the Hdh IGFBP7 gene was 1519-bp long with an open reading frame of 720-bp corresponding to a putative polypeptide of 239 amino acids. The molecular mass of its mature protein was approximately 23.44 KDa with an estimated isoelectric point (pI) of 5.35, and it shared significant homology with IGFBP7 gene of H. madaka. Hdh IGFBP7 has a characteristic IGFBP N-terminal domain (22&ndash, 89 aa), a kazal-type serine proteinase inhibitor domain (77&ndash, 128), and an immunoglobulin-like C2 domain (144&ndash, 223). Furthermore, twelve cysteine residues and a signature motif of IGFBPs (XCGCCXXC) were found in its N-terminal domain. Phylogenetic analysis revealed that Hdh IGFBP7 was aligned with IGFBP7 of H. madaka. Tissue distribution analysis showed that the mRNA of Hdh IGFBP7 was expressed in all examined tissues, with the highest expression level observed in the mantle and gill tissues. The expression level of Hdh IGFBP7 mRNA was relatively higher at the juvenile stage during its metamorphosis period. In situ hybridization showed that Hdh IGFBP7 transcript was expressed in epithelial cells of the dorsal mantle pallial and mucus cells of the branchial epithelium in gill. These results provide basic information for future studies on the role of IGFBP7 in IGF regulation of shell growth, development and metamorphosis of abalone.

Carbonic anhydrase VI (CA VI) has been characterized as a secretory isozyme in mammals. Our present study confirmed the occurrence of CA VI in pufferfish ( Takifugu rubripes ). In this study, genomic sequence information for the CA VI of pufferfish was used for molecular cloning. We cloned a 1821 bp cDNA sequence, which consisted of a complete coding sequence of 1623 bp and a deduced amino acid sequence of 540 amino acids from the open reading frame. A BLAST search indicated that this protein exhibits 53%, 79%, and 67% identity with human, tilapia, and gar CA VI, respectively. It also shows 63%–77% identity with other fish CA VI-like sequences (zebrafish, Asian arowana, salmon, and large yellow croaker). Moreover, alignment of two or more sequences revealed that the protein sequence of pufferfish CA VI has 34%–37% identity with mammalian and fish CA II sequences. An NH 2 -terminal signal peptide of 18 amino acids in length was predicted in the pufferfish CA VI sequence. Three potential N-linked glycosylation sites and two cysteine residues (Cys-28 and Cys-209) that are likely to form one disulfide bond were present in pufferfish CA VI. In silico and phylogenetic analyses revealed that pufferfish CA VI is an extracellular secretory protein. Active site analysis indicated that this protein is a low-activity CA isozymes due to a characteristic Val/Ile substitution at position 207. Homology modeling of puffer CA VI was performed using the crystal structure of human carbonic anhydrase XIV as a template structure, based on high similarity. Reverse transcription-polymerase chain reaction (PCR), quantitative PCR and in situ hybridization results revealed that, the pufferfish CA VI is highly expressed in liver tissue.

In this study, an experiment was conducted to investigate the annual reproductive cycle of a Korean flat bittering, Acheilognathus rhombeus, from Ogok-myeon located in Seomjin River. The reproductive cycle was examined histologically regarding water temperature and day length of the habitat, the gonadosomatic index (GSI), and developmental characteristics of female and male gonads. The maximum GSI was found to be 3.50±0.53 and 1.36±0.14 for females and males, respectively, when the water temperature and day light was 16.9°C and 11.3 hours, respectively in October 2018. On the other hand, the minimum GSI was found to be 0.16±0.09 and 0.69±0.15 for males and females in December 2018 and February 2019, respectively. The ovipositor of females appeared from August to November 2018. We compared and calculated the stages of germ cell developmental characteristics in the testis and ovaries to determine the reproductive cycle. According to the result, we classified the female A. rhombeus reproductive cycle into four phases, which are ripe and spawning phase (October), degenerative phase (November to December), growing phase (January to March) and mature phase (April to September). The annual reproductive cycle of male A. rhombeus was categorized into four phases: mature phase (June to October), degenerative phase (November to March), resting phase (April) and growing phase (May). The Korean flat bittering is an autumn-spawner as the main spawning season in October. In male, testicular spermatogonia appeared all year-round, and the ripe and releasing phase, which is characteristics of the spawning season in other fish, did not appear.

In this present study, we examined the anti-inflammatory and anti-oxidative properties of alcoholic lemon myrtle extract (LME). The total polyphenol and flavonoid content of LME were determined as 118.77 and 14.53 mg/g extract, respectively. LME showed anti-oxidative properties, such as 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) and 2,2′-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS) radical scavenging activity. The anti-inflammatory activities of LME were investigated using the lipopolysaccharide-stimulated murine macrophage RAW 264.7 cells. Pretreatment with LME was performed at non-cytotoxic concentrations of 10–100 μg/mL. LME inhibited the production of inflammatory mediators such as nitric oxide (NO). Enzyme-linked immunosorbent assay and reverse-transcriptase polymerase chain reaction (RT-PCR) revealed that pretreatment with LME suppressed the protein expression and mRNA levels of pro-inflammatory cytokines such as interleukin IL-6, and tumor necrosis factor (TNF)-α in a concentration-dependent manner, respectively. These results suggest that LME could be used as a potential therapeutic agent having potent anti-inflammatory effects that could be used to treat inflammatory bowel disease.

Pacific abalone (Haliotis discus hannai) is a highly commercial seafood in Southeast Asia. The aim of the present study was to improve the sperm cryopreservation technique for this valuable species using an antifreeze protein III (AFPIII). Post-thaw sperm quality parameters including motility, acrosome integrity (AI), plasma membrane integrity (PMI), mitochondrial membrane potential (MMP), DNA integrity, fertility, hatchability, and mRNA abundance level of heat shock protein 90 (HSP90) were determined to ensure improvement of the cryopreservation technique. Post-thaw motility of sperm cryopreserved with AFPIII at 10 µg/mL combined with 8% dimethyl sulfoxide (DMSO) (61.3 ± 2.7%), 8% ethylene glycol (EG) (54.3 ± 3.3%), 6% propylene glycol (PG) (36.6 ± 2.6%), or 2% glycerol (GLY) (51.7 ± 3.0%) was significantly improved than that of sperm cryopreserved without AFPIII. Post-thaw motility of sperm cryopreserved with 2% MeOH and 1 µg/mL of AFPIII was also improved than that of sperm cryopreserved without AFPIII. A combination of 10 µg/mL AFPIII with 8% DMSO resulted in the highest post-thaw motility, showing AI of 60.1 ± 3.9%, PMI of 67.2 ± 4.0%, and MMP of 59.1 ± 4.3%. DNA integrity of sperm cryopreserved using 10 µg/mL AFPIII combined with 8% DMSO was not significantly (p &gt, 0.05) different from that of fresh sperm. Cryopreservation using a combination of AFPIII with 8% DMSO improved fertilization and hatching rates of sperm compared to that of cryopreservation without supplementation of 10 µg/mL AFPIII. Sperm cryopreserved using AFPIII showed higher mRNA abundance levels of HSP90 than those cryopreserved without AFPIII. Results of the present study suggest that 10 µg/mL AFPIII combined with 8% DMSO can be used for large scale cryopreservation of Pacific abalone sperm and for hatchery production.

Carbonic anhydrase (CA), a ubiquitous enzyme found in many species, including fishes, is involved in physiological functions such as pH homeostasis, calcification, photosynthesis, and ionic regulation. CA I, a member of the α-CA family, is a cytoplasmic isozyme involved in carbon dioxide transport, ion exchange, and acid-base balance. Approximately half of the extant shark species occur only in deep waters; however, few published studies on sharks include these taxa. As fisheries worldwide enter deeper waters, the provision of biological data for these little-known taxa is critical to their management and conservation. To address this limitation, we aimed to detect CA I in various tissues of the shortspine spurdog (Squalus mitsukurii) and characterize its physicochemical properties by using sodium dodecyl-sulfate polyacrylamide gel electrophoresis and isoelectric focusing, together with immunohistochemistry. CA I was detected on SDS-PAGE and western blot analysis as a specific band at 29 kDa in various tissues of the shortspine spurdog, and as a specific band at pI 6.5 in various tissues of the shortspine spurdog by IEF and western blot analysis. CA I immunoreactivity in various tissues of the shortspine spurdog was detected in intracellular locations. To our knowledge, this is the first report of the localization of CA isozymes in various tissues of S. mitsukurii.

Carbonic anhydrase (CA) is a key element for maintaining acid base balance in fish. In our present experiment, novel CA isozymes were identified from the pear puffer (Takifugu vermicularis). Based on the high homology of two predicted CA sequences of the tiger puffer (Takifugu rubripes), a 1715bp novel cDNA was obtained from T. vermicularis. The open reading frame showed a complete coding sequence of 552bp with a deduced peptide sequence of 183 amino acids that exhibited highest (97%) identity with pufferfish putative CA III and CA IV-like sequences. In addition, this translated protein sequence showed 36-37% identity with zebrafish CA IV-like, CA XVa, CA XVb, and CA XVc proteins. Phylogenetic analysis revealed that the pufferfish novel protein (pCAn) was a membrane-bound CA protein. Alignment of multiple CA sequences illustrated that most of the putative active site residues of the pCAn isozyme were situated at highly conserved regions of the CA sequences. Examination of motif distribution suggested that the pCAn isozyme was very similar to the puffer predicted CA IV-like isozyme. Reverse transcription-polymerase chain reaction (PCR) analysis showed highly differential expression in the brain, gills, kidney, and muscle, whereas CA mRNA expression was almost absent in heart, liver, and intestine. Quantitative PCR expression of CA mRNA abundance suggested several-fold higher expression of pCAn isozymes in the gills compared to other tissues tested. Our results suggest that the pCAn isozyme might be related to CA IV-like isozymes. Further functional studies are needed to investigate the function of the pCAn isozyme in T. vermicularis.

The peel of pink-colored tomato is transparent due to the lack of accumulation of the flavonoid naringenin chalcone during ripening. A strong correlation was found between flavonoid expression and the function of SlMYB12, which is a transcriptional regulator of flavonoid biosynthesis. Thus, SlMYB12 is a strong candidate gene underlying the pink phenotype. Three allelic variants, a 603 bp deletion, a nucleotide substitution (C > T), and a 1 bp insertion (TG > TAG) in the SlMYB12 gene have been previously reported. We performed PCR genotyping based on these three allelic variations in 47 tomato cultivars displaying either a pink or red phenotype. However, the genotype did not match with the expected phenotype in one pink cultivar “Prime Alexander”. This cultivar was therefore self-pollinated to produce 20 progeny plants. To identify new mutations in SlMYB12, the sequence of genomic DNA and CDS were compared between the progeny 17 and the reference line, Heinz 1706. A novel G > T nucleotide substitution was found in the 2nd intron. This SNP leads to a deletion of 7 bp (GTAACAG) from the end of the 2nd exon, resulting in a premature stop codon. The presence of this SNP associates the pink phenotype with the genotype. This novel SNP will be useful as a genetic marker for marker-assisted breeding of pink tomato.

Global population is increasing day-by-day, simultaneously, crop production need to increase proportionately. Whereas, increase crop production being restricted due to abiotic and biotic stresses. Abiotic stresses are adversely affected crop growth and development, leading to crop loss globally and thereby causing huge amount of economic loss as well. Contrary, pathogens are attacked the plants imposing biotic stress and severely hampers the yield. Therefore, it is prime need to understand the molecular mechanism and genes involved to minimize the biotic and abiotic stresses for mitigating the Brassica vegetable crop losses. The stress responsive, pathogens related genes are involved in tolerance or resistance to stress in plants that are cross-talk with different types of stress components in signal transduction pathways. The plants have their own mechanism to overcome biotic and abiotic stresses to follow the abscisic acid (ABA)-dependent and ABA-independent pathways. Several transcription factors such as WRKY, Alfin-like, MYB, NAC, DREB, CBF are integrating to various stress signals and controlling the gene expression through networking with their related cis-elements. To develop stress tolerance and/or resistant crops plants, there is need to realize both of the plant and pathogenic disease development mechanisms. Therefore, this article is focused on (i) major and devastating stresses on vegetable crops, (ii) role of genes to overcome the stresses, and (iii) differential genes expressed under biotic and abiotic stresses in Brassica oleracea and B. rapa for getting insight of the mechanisms of development of resistance lines.

Gonadotropin-releasing hormone (GnRH) is a key hypothalamic neuropeptide regulating the reproductive function of vertebrates and invertebrates. In the present study, we report the role of GnRH in gamete cell proliferation of Pacific abalone, Haliotis discus hannai using BrDU incorporation, and analyzed the expression of reproductive responsive genes. In vivo injection of buserelin and H. discus hannai GnRH peptides (GnRH-I and GnRH-II) at doses of 250 and 500 ng/g BW resulted in a significant increases in the number of testicular and ovarian proliferating cells. Administration of GnRH peptide at higher doses was less effective in stimulating gonadal cell proliferation. A qPCR analysis revealed that the mRNA expression of GnRH, GnRH-R, and serotonin-R was elevated in the male gonad compared with female gonad in all treatment groups. Compared with control and other treatment groups, the expression profile of GnRH, GnRH-R, and serotonin-R was significantly higher after treatment with buserelin and synthetic GnRH peptides at doses of 250 ng/g BW in both male and female gonad. However, the gonadal tissues expressed lower levels of mRNA following the treatment with GnRH peptide at the higher dose (1000 ng/g BW). Furthermore, the results of comet assay demonstrated that gonadal cells did not show significant differences in the average level of fragmented DNA and tail length between control and treated groups. Taken together, these findings suggest that administration of GnRH peptide at a concentration of 250 ng/g BW effectively accelerated the gametogenesis of abalone. This strategy will enhance the benefits of breeding technology in this species, or may useful for increasing the aquaculture production.

Gonadotropin-releasing hormone (GnRH) is a key neuropeptide of vertebrates, involved in gonadal maturation and primarily regulated by the hypothalamic-pituitary-gonadal axis. The full length GnRH-like cDNA isolated from the cerebral ganglion of Haliotis discus hannai, encodes a deduced protein of 216 amino acids with a theoretical molecular mass of 23.36 kDa and an isoelectric point of 7.72. The GnRH transcript comprises a putative signal peptide, a GnRH dodecapeptide, a proteolytic processing site, and a GnRH associated peptide (GAP). Comparative structural analysis revealed that the cloned sequence shares relatively high homology with other molluscan species and a lower degree of amino acid identity with GnRH of piscine vertebrates. Phylogenetic comparison with other known GnRH genes revealed that the GnRH-like mRNA of H. discus hannai is most closely related to the marine gastropod, H. laevigata. Three-dimensional homology structure of H. discus hannai GnRH and GAP exhibited a helix-loop-helix structure. Quantitative PCR assay demonstrated wide expression of GnRH in all ganglia, among them cerebral ganglion exhibited the highest expression level. Significantly higher expression was observed in cerebral ganglion and gonadal tissues at higher effective accumulative temperature (1000 °C). In situ hybridization showed that the GnRH expressing neurosecretory cells distributed throughout the cortex of the cerebral ganglion. These results suggest that GnRH synthesized from the cerebral ganglia may be involved in gonadal maturation and regulating the secretion of other reproductive hormones.

In this study, an 1888-bp carbonic anhydrase XII (CA XII) sequence was cloned from the brain of the pufferfish, Takifugu rubripes. The cloned sequence contained a coding region of 1470-bp, which was predicted to translate into a protein of 490 amino acid residues. The predicted protein showed between 68–56% identity with the large yellow croaker (Larimichthys crocea), tilapia (Oreochromis niloticus), and Asian arowana (Scleropages formosus) CA XII proteins. It also exhibited 36% and 53% identity with human CA II and CA XII, respectively. The cloned sequence contained a 22 amino acid NH2-terminal signal sequence and three Asn-Xaa-Ser/Thr sequons, among which one was potentially glycosylated. Four cysteine residues were also identified (Cys-21, Cys-201, Cys-355, and Cys-358), two of which (Cys-21 and Cys-201) could potentially form a disulfide bond. A 22-amino acid COOH-terminal cytoplasmic tail containing a potential site for phosphorylation by protein kinase A was also found. The cloned sequence might be a transmembrane protein, as predicted from in silico and phylogenetic analyses. The active site analysis of the predicted protein showed that its active site residues were highly conserved with tilapia CA XII protein. Homology modeling of the pufferfish CA XII was done using the crystal structure of the extracellular domain of human carbonic anhydrase XII at 1.55 Å resolution as a template. Semi-quantitative reverse transcription (RT)-PCR, quantitative PCR (q-PCR), and in situ hybridization confirmed that pufferfish CA XII is highly expressed in the brain.