Hepatocyte nuclear factor 4 alpha (HNF4α) plays a critical role in hepatic lipid metabolism in mammals, particularly with respect to fatty acid β-oxidation (FAO). However, its gene structure and biological function in fish remain largely unknown. In this study, the hnf4α gene, which encodes a peptide consisting of 454 amino acids, was characterized from the tiger puffer (Takifugu rubripes). As a member of the nuclear receptor superfamily, the Hnf4α protein contains a conserved deoxyribonucleic acid-binding domain at the N-terminus and a C-terminal multifunctional hydrophobic ligand-binding domain, which binds fatty acids. Amino acid sequence alignment, gene structure comparison, and phylogenetic analysis indicated that tiger puffer Hnf4α is highly conserved relative to mammals and other teleosts, suggesting evolutionarily conserved functions. Tissue distribution analysis indicated that hnf4α transcript levels were highest in the intestine, followed by the liver and kidney. To explore whether HNF4α participates in regulating hepatic FAO in tiger puffer, a lipid accumulation model was first established in hepatocytes using 200 μM oleic acid (OA). Subsequent treatment with 100 μM docosahexaenoic acid (DHA) visually diminished OA-induced lipid droplets accumulation, accompanied by a significant reduction in triglyceride content and upregulation of hnf4α as well as several FAO-related genes, including peroxisome proliferator-activated receptor alpha (pparα), peroxisome proliferator-activated receptor γ coactivator 1 alpha (pgc1α), and carnitine palmitoyltransferase 1ab (cpt1ab). Taken together, these findings offer initial evidence that Hnf4α may be involved in the DHA-induced promotion of FAO in tiger puffer hepatocytes.
Dopamine (DA) regulates diverse physiological processes via dopamine receptors (DRs), yet the molecular activity and physiological relevance of dopamine receptor D4 paralogs in teleosts remain unclear. Here, we identified and characterized Drd4b (LcDrd4b) from Larimichthys crocea. Sequence and phylogenetic analyses supported its assignment to the DRD4 lineage. Heterologous expression in human embryonic kidney (HEK293) cells showed that LcDrd4b exhibited membrane-associated localization and DA-associated redistribution, reduced forskolin-stimulated cyclic adenosine monophosphate (cAMP) accumulation, and activated extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. Gene expression profiling revealed relatively high Lcdrd4b expression in the intestine at early reproductive stages and prominent gonadal expression at stage IV, especially in mature testes. Tyrosine hydroxylase (Lcth) showed high hepatic expression at stages II-III and a sex-related shift at stage IV. Together, these findings establish LcDrd4b as a functional DR in a marine teleost and provide a molecular basis for further studies of DA signaling in teleost physiology.
Elongation of very long-chain fatty acids (Elovl) proteins play essential roles in the biosynthesis of long-chain polyunsaturated fatty acids (LC-PUFAs). However, endogenous LC-PUFA biosynthesis in crustaceans remains incompletely understood, partly due to limited functional characterization of elongases. In this study, we cloned and functionally characterized an elovl1/7-like gene from the red swamp crayfish (Procambarus clarkii). The full-length cDNA contains an open reading frame of 1155 bp encoding a 384-amino acid protein with five predicted transmembrane domains and a conserved histidine box (HXXHH). Phylogenetic analysis showed that P. clarkii Elovl1/7-like clusters with crustacean homologs, forming a distinct clade separate from vertebrate Elovl1 and Elovl7. Tissue expression analysis revealed ubiquitous expression, with the highest levels in the hepatopancreas and gill. Functional characterization using a heterologous yeast expression system demonstrated that P. clarkii Elovl1/7-like catalyzed the elongation of C18:1n-9 to C20:1n-9 and converted C18:2n-6 and C18:3n-3 to their corresponding C20 products, while no elongation activity was detected toward C20 or C22 polyunsaturated fatty acid substrates. These results indicate that the enzyme preferentially acts on C18 fatty acids and has a limited role in elongating longer-chain substrates. This study provides functional evidence for the involvement of Elovl1/7-like in fatty acid elongation in P. clarkii and contributes to understanding lipid metabolism in crustaceans.
The remarkable diversity of decapod crustaceans inhabiting a wide range of osmotic environments reflects their evolutionary success, likely driven by the variety of mechanisms for body fluid regulation involving both molecular and systemic adaptations. In recent decades, considerable research has focused on the osmoregulatory strategies of decapod crustaceans occupying distinct osmotic niches. However, limited information is available on the effects of salinity on the activity and expression of gill (Na+, K+)-ATPase in terrestrial and semi-terrestrial species. This study investigates key aspects of osmoregulation in the semiterrestrial crab Goniopsis cruentata during acclimation to varying salinity levels. After exposure to 10, 20, 30, and 40 ‰S for 5 days, the crabs exhibited strong hyper- and hypo-osmoregulatory capabilities, reaching isosmotic conditions at approximately 30 ‰S. The Total Regulatory Capacity, expressed as a dimensionless ratio of hemolymph to external osmolality variation, was 0.08, with hyper- and hypo-osmoregulatory values of 0.06 and 0.12, respectively. Goniopsis cruentata demonstrated increased mRNA expression of (Na+, K+)-ATPase under hypo-osmotic conditions, despite a concurrent reduction in enzyme activity. These findings suggest that both hyper- and hypo-osmoregulatory mechanisms in G. cruentata are dependent only minimally on branchial (Na+, K+)-ATPase activity and instead rely more strongly on other ion transporters to maintain body fluid homeostasis. These results contrast with observations in aquatic crabs, in which branchial (Na+, K+)-ATPase plays a central role in osmoregulation.
In the present study, myoblast determination protein (myod) and myogenic factor 5 (myf5), two critical primary myogenic regulatory factors (MRFs) that govern muscle growth were characterized in snow trout (Schizothorax richardsonii). In addition, their mRNA expression was examined in relation to age, sex, nutritional conditions and temperature regimes. The full-length of Srmyod and Srmyf5 mRNA sequences were 1638 and 1397 nucleotides, respectively; with open reading frames of 825 and 723 nucleotides, which encoded proteins of 273 and 240 amino acid residues. In silico protein-protein interaction, nuclear localization signals, ligand-binding residues and tertiary protein structures indicated their putative roles in dimerization, DNA binding, and transcriptional activation. With respect to muscle mRNA expression, Srmyod was upregulated in older fish (2+ age) and conversely Srmyf5 was higher in younger fish (0+ age). There were no sex-dependent differences in MRFs expression. Three weeks of refeeding following three weeks of starvation significantly upregulated the transcriptional levels of both MRFs, corroborating the compensatory growth potential of the fish. The expression of Srmyf5 and Srmyod were concurrently high in fish fed 35% dietary protein. In contrast, dietary lipid levels (3-13%) had no significant effect on MRF expression. With respect to rearing temperature (6-24 °C), Srmyf5 expression showed marked upregulation at 18 °C, consistent with optimal fish growth. Overall, these findings reveal the conserved molecular profile and distinct transcriptional regulation of myod and myf5 in snow trout, under different biological, nutritional and environmental determinants of growth.
Honeybees (Apis mellifera L., 1758) are a valuable model for aging research due to their pronounced phenotypic plasticity, whereby genetically similar worker bees exhibit marked lifespan differences shaped by environmental and seasonal factors. We investigated age-related changes in selected epigenetic mechanisms-DNA 5-methylcytosine (5-mC), histone modifications-and autophagy-related gene expression in summer and winter worker bees originating from three independent hives. Bees were sampled as newly emerged individuals and at middle and late life stages, corresponding to two- and four-week-old summer bees and two- and four-month-old winter bees. Global DNA cytosine methylation (5-mC) levels did not vary with age in summer and winter bee generations. In contrast, DNA methyltransferases (DNMTs) exhibited pronounced tissue- and age-dependent expression patterns, including strong upregulation of DNMT isoform 3 gene (Dnmt3) in older abdominal tissues and increased expression of maintenance DNMTs, suggesting locus-specific regulatory changes. Examined histone H3 methylation marks showed no age-related variation. However, histone H3 acetylation at lysine residues 18 and 27 (H3K18ac and H3K27ac) displayed clear seasonal differences: they remained unchanged in summer bees but were elevated in winter bees, particularly at middle age. While whole-body histone acetyltransferase (HAT) activity did not differ among groups, expression of the longevity-associated deacetylase, sirtuin1 (SIRT1), declined with age in winter bees but increased in summer bees. Finally, autophagy-related genes were predominantly upregulated with age in summer bees and downregulated in winter bees. These findings demonstrate that honeybee aging can be influenced by seasonally modulated epigenetic and autophagy-related mechanisms.
Low-fishmeal diets often impair feed utilization and disturb physiological homeostasis in carnivorous fish, and supplementation with umami-related amino acids may help counteract these effects. This study evaluated the effects of dietary alanine or glycine supplementation in largemouth bass (Micropterus salmoides) fed low-fishmeal diets. Seven isonitrogenous and isolipidic diets were formulated, including a low-fishmeal control diet and six diets supplemented with alanine or glycine at 0.5%, 1.0%, or 2.0%. Juvenile fish (initial body weight 4.60 ± 0.01 g) were fed these diets for 8 weeks. Compared with the control, alanine or glycine supplementation increased weight gain and specific growth rate, reduced feed conversion ratio, and decreased feeding rate. The most consistent improvements were observed with 1.0% alanine and 0.5% glycine. Moderate supplementation also improved intestinal villus morphology and was associated with more favorable hepatic biochemical and redox profiles. In the liver, alanine or glycine elevated superoxide dismutase and catalase activities and reduced malondialdehyde content, indicating improved redox status. These biochemical changes were accompanied by transcriptional responses consistent with enhanced antioxidant regulation and attenuated inflammatory signaling, including increased nuclear factor erythroid 2-related factor 2 (nrf2) and catalase (cat), decreased Kelch-like ECH-associated protein 1 (keap1) in the most responsive groups, downregulated nuclear factor kappa B (nf-κb) and interleukin-1 beta (il-1β), and upregulated interleukin-10 (il-10). Although appetite-related genes also responded to supplementation, these changes were not associated with increased feeding rate. Collectively, dietary alanine or glycine improved feed utilization and growth performance, together with coordinated gut-liver physiological responses, without a corresponding increase in feeding rate.
Fruitless (fru) is a conserved transcriptional regulator of sex-specific traits and mating behavior in insects; however, its functions in crustaceans remain largely unknown. In the brine shrimp Artemia franciscana, we identified and characterized a novel fru homolog, which we named Afrfru. The Afrfru gene encodes a BTB-C2H2 zinc-finger transcription factor. Phylogenetic analysis placed the Afrfru protein (AfrFRU) in close proximity to other branchiopod fru homologs. Expression profiling revealed predominant expression in the second antenna of males, while transcript levels peaked during middle oogenesis in females. Knockdown of Afrfru via RNA interference (RNAi) in females disrupted oocyte development and led to abnormal oocyte accumulation. In males, Afrfru RNAi knockdown reduced locomotor velocity and markedly decreased pairing success. To investigate downstream mechanisms, we conducted sex-specific RNA-seq after Afrfru-RNAi. In knockdown females, differentially expressed genes (DEGs) were enriched in glutathione and arachidonic acid metabolism pathways; expression of genes associated with insect hormone biosynthesis and ecdysone synthesis was upregulated. In Afrfru-RNAi males, DEGs predominantly exhibited downregulated expression, particularly among genes involved in cytochrome P450-mediated xenobiotic/drug metabolism, glutathione metabolism, and fatty acid metabolic processes. Together, these results indicate that Afrfru contributed to reproductive maturation and pairing behavior in A. franciscana, likely through sex-specific metabolic and hormonal programs. Our study expands the understanding of reproductive behavior regulation in Artemia and extends the known functional scope of fru beyond insect models.
Carboxylesterases (CarEs) are key phase I detoxification enzymes that either directly metabolize insecticides or sequester insecticide molecules, thereby reducing their toxicity. Previous studies demonstrated that the A127D mutation in carboxylesterase 001C (CarE001C) of Helicoverpa armigera significantly enhanced hydrolase activity toward the model organophosphate substrate diethyl 4-methylumbelliferyl phosphate (dEUP). To further explore the functional implications of this mutation, six CarE001C variants containing multiple site mutations were generated using the CarE001CA127D mutant as a template through a multiple site-directed mutagenesis approach. The recombinant proteins were successfully expressed in Escherichia coli, purified, and confirmed by SDS-PAGE and Western blot analyses. Enzyme kinetics assays revealed that all multiple-site mutants exhibited significantly reduced esterase activity toward α-naphthyl acetate compared with the wild-type CarE001C. In vitro metabolism assays indicated that several of these mutants exhibited improved hydrolase activity toward specific insecticides. Among them, three mutants (OP4, OP5, and OP6) showed elevated hydrolase activities toward chlorpyrifos, with specific activities ranging from 26.3 to 38.8 nmol·min-1·mg protein-1. Additionally, two mutants demonstrated increased detoxification capacities for beta-cypermethrin and methomyl, exhibiting specific activities of 13.2 and 1.7 nmol·min-1·mg protein-1, respectively. Collectively, these results suggest that multiple site mutations can significantly alter the catalytic properties of H. armigera CarE001C, leading to qualitative changes in insecticide hydrolase activity. This work provides new insights into the adaptive potential of insect esterases in insecticide detoxification, thereby deepening our understanding of their roles in resistance evolution and potential applications in bioremediation.
Serine peptidases (SP) are crucial proteolytic enzymes involved in food digestion and other processes, including participation in the regulation of signal transduction in response to developmental or pathological cues. This study presents a genome-wide investigation of SPs comprising less than 300 amino acid residues and containing a single catalytic peptidase domain in the yellow mealworm Tenebrio molitor (L.), a globally distributed species that infests stored cereals and other food products. Detailed analyses of gene structures (exon-intron pattern), chromosomal localization, and transcriptional orientation revealed numerous SP-encoding loci spread across nine chromosomes of the T. molitor genome. Most of these genes are organized in tandem arrays, forming clusters of closely related genes, thus suggesting that the SP gene family has undergone substantial expansion through gene duplication events. Phylogenetic gene family analysis further uncovered potential displaced duplications resulting from intra- and/or inter-chromosomal gene movements. Gene expression profiling across developmental stages revealed diverse expression patterns, pointing to previously unrecognized physiological roles of single domain SPs beyond their well-established function in digestion. Overall, this study provides a comprehensive resource on insect SP genes and their developmental expression profiles.
This study investigated the physiological and metabolic responses of juvenile Chinese mitten crab (Eriocheir sinensis) to dietary inclusion of coenzymatically hydrolyzed anchovy-cottonseed protein (HACP) with or without arginine and methionine supplementation. A 56-day feeding trial was conducted. Juvenile crabs were fed a control diet containing 15% fishmeal and experimental diets in which fishmeal was partially replaced by HACP at graded levels (5%, 10%, and 15%). Increasing dietary HACP levels significantly affected growth performance, feed intake, digestive enzyme activities, hepatopancreatic condition, and intestinal microbial composition. The 5% HACP inclusion maintained growth performance and protein utilization comparable to the control, whereas higher inclusion levels reduced feed intake and digestive enzyme activity, suggesting potential alterations in digestive enzyme activity and metabolic status. Arginine and methionine supplementation partially alleviated these effects by enhancing trypsin and lipase activities, reducing hepatopancreatic stress indicators, and improving antioxidant status, suggesting potential improvements in nutrient utilization and metabolic responses. Microbiota and metabolomic analyses further revealed coordinated shifts in intestinal microbial composition and metabolic pathways associated with amino acid, lipid, and energy metabolism. Overall, these findings indicate that dietary HACP influences physiological function through integrated effects on digestion, metabolism, and microbiome-metabolome interactions, and highlight the role of amino acid balance in mediating metabolic response under altered dietary protein composition.
The multifunctionality of hemocyanins is intrinsically linked to their molecular heterogeneity. The expression levels of different hemocyanin subunits can change in response to environmental influences and during ontogenetic processes. In this work, we identified a number of differentially expressed isoforms of hemocyanin from the hemolymph of red king crab Paralithodes camtschaticus of the Western Sakhalin population at different stages of the molting cycle using proteomics informed by transcriptomics. Determination of the quaternary structure of hemocyanin at different stages of the molting cycle using size-exclusion chromatography and dynamic light scattering showed that the variability of the subunit composition did not significantly affect the oligomerization propensity of the protein. Differential scanning calorimetry was performed with subunits and heterohexamers of hemocyanin at different stages of the molting cycle. Native heterohexamers showed a significant similarity in their thermal denaturation behavior which could be interpreted in terms of a two-state model. Based on the bioinformatics-assisted analysis of hemocyanin isoforms sequences we assume that the rate-limiting step in the denaturation process of the native heterohexamers is the unfolding of cooperative thermodynamic domains formed through interactions between highly conserved residues from the central domains of the subunits. Thus, the variability in the isoform composition of native oligomers of Paralithodes camtschaticus hemocyanin during the molting cycle, does not significantly affect their quaternary structure or conformational stability due to the features of their molecular architecture.
Vitamin C (VC) plays a crucial role in regulating redox homeostasis; however, its biochemical responses in Penaeus vannamei to graded dietary levels, particularly in relation to immune, antioxidant defense and structural protein metabolism remain insufficiently characterized. This study therefore investigated the physiological and biochemical modulations and VC requirement of P. vannamei by evaluating the effects of graded vitamin C concentrations (5.99-157.78 mg/kg) on growth, immune responses, hepatopancreas vitamin C concentration and muscle hydroxyproline and collagen concentrations. Six diets were formulated to contain VC at levels of 0, 30, 60, 90, 120 and 150 mg/kg (designated as C0, C30, C60, C90, C120 and C150, respectively). Quadruplicate groups of 20 shrimp (0.70 ± 0.01 g) were fed the diets for 49 days. Growth was significantly increased in shrimp fed C60, C90, C120 and C150 diets compared to the C0 group. Superoxide dismutase activity was significantly increased in C60, C90 and C120 groups in hemolymph, and significantly increased in C60, C90, C120 and C150 groups in hepatopancreas compared to the C0 group. Catalase activity and total antioxidant capacity were significantly increased in shrimp fed C90 diet compared to the C0 group. Hepatopancreas VC and muscle hydroxyproline and collagen concentrations were gradually increased with the increment in dietary VC concentrations (up to 63.1 mg/kg). Based on broken-line regression and second-order polynomial regression models in relation to growth and muscle hydroxyproline, the estimated optimum and maximum VC requirements varied from 77.8 to 151.7 mg/kg. These findings demonstrate that VC is a physiological modulator of redox balance and hydroxyproline synthesis of shrimp.
Fipronil, a widely used phenylpyrazole insecticide, is frequently detected in aquatic environments, where it induces neurotoxicity, oxidative stress, and metabolic disruption in non-target species. In light of these ecological concerns, treatment strategies employing exogenous amino acids and their derivatives have emerged as promising protective approaches. However, metabolomics-based assessments of such interventions remain scarce. This study investigated the protective potential of 5-aminolevulinic acid (5-ALA), a heme biosynthesis precursor known to support mitochondrial function, against fipronil-induced toxicity in adult zebrafish (Danio rerio) through NMR-based metabolomic profiling. Metabolic profiling showed that a 72-h co-exposure with 5-ALA (0.1 mM) was associated with attenuation of the effects of fipronil (0.46 μM) on neurotransmitter metabolism and energy metabolism. These findings suggest that the mitigating effects of 5-ALA may involve modulation of GABAergic signaling, potentially counteracting fipronil-related excitotoxic stress. In addition, 5-ALA partially relieved fipronil-induced disruptions in energy metabolism. Overall, our results suggest that 5-ALA may act as a metabolic modulator capable of attenuating fipronil-induced neurotoxicity and associated disruptions in energy metabolism in zebrafish. Although this study offers mechanistic insights at the metabolic level, additional focused biochemical and receptor-level studies in environmentally-relevant settings are necessary to confirm these findings.
Dual-specificity protein phosphatase 4 (DUSP4), a critical negative regulator of the mitogen-activated protein kinase (MAPK) signaling pathway, enters the nucleus via conserved nuclear localization sequences (NLS) to dephosphorylate key kinases in vertebrates. However, the functional and evolutionary significance of these NLSs in invertebrates remains unclear. We investigated the effects of the typical DUSP4 NLS RRRAK and KKRx10KQRR on the subcellular localization and phosphatase function of the CgDUSP4 protein in the Pacific oyster (Crassostrea gigas). Comparative protein sequence analysis revealed that CgDUSP4 lacks the conserved NLSs. The mutant CgDUSP4 (mCgDUSP4) with typical NLSs was generated to investigate the effects of NLS on the subcellular localization and function of CgDUSP4. Notably, although mCgDUSP4 was not completely localized to the nucleus, it endowed mCgDUSP4 with the novel ability to specifically dephosphorylate p38 and JUN N-terminal kinase (JNK). This reveals that substrate recognition and nuclear import are separable functions encoded within these motifs. These findings provide mechanistic insights into the molecular evolution of DUSP4 and contribute to understanding how structural innovations in regulatory proteins shape the diversification of signaling pathways across metazoans.
Fertility decline is common in interspecific hybrids, as observed in the F1 hybrids between the bay scallop (Argopecten irradians irradians) and Peruvian scallop (A. purpuratus). While it is known that mitochondria may regulate fertility, their role in hybrid fertility decline remains unclear. This study investigated mitochondrial ultrastructure, regulatory gene expression, and function in relation to fertility decline in F1 hybrids. Fertility assessment revealed significant differences in fertilization rates at 30 min after fertilization between the low fertility group (Group L), the high fertility group (Group H), and the control group (self-crosses of Ai, Group N). Group L exhibited significant mitochondrial abnormalities, such as swelling, matrix vacuolization, compared to Group H and Group N. Gene expression analysis revealed significant alteration in mitochondrial regulation in hybrids, particularly the down-regulation of MFN2 (mitofusin 2) and NRF1 (nuclear respiratory factor 1) and up-regulation of DRP1 (dynamin-related protein 1), especially in Group L. Additionally, the expression of the autophagy gene ATG5 (autophagy-related gene 5) was up-regulated in Group H and down-regulated in Group L. ATP6 (ATP synthase gene) and other electron transport chain genes were significantly down-regulated in Group L. The antioxidant defense gene SOD2 (superoxide dismutase 2) expression increased in Group H but significantly decreased in Group L. Functionally, hybrids showed reduced mitochondrial membrane potential, disrupted calcium ion homeostasis, increased membrane permeability, and decreased ATP production, with the most severe impairments in Group L. These findings emphasize the possible key role of mitochondrial in the hybrid infertility of marine bivalves.
Kisspeptin is a conserved neuropeptide that regulates reproductive function and other physiological processes across vertebrates via its cognate G protein-coupled receptors (GPCRs), commonly referred to as kisspeptin receptors (KissRs). In teleosts, extensive gene duplication and lineage-specific diversification have generated multiple kisspeptin ligands and receptors, yet the molecular properties and signaling profiles of individual KissRs remain incompletely characterized. Here, we identified and cloned a Kissr3-type receptor from the large yellow croaker (Larimichthys crocea), designated LcKissr3. Sequence and phylogenetic analyses classified LcKissr3 within the teleost Kissr3 clade. Functional assays in a human embryonic kidney cell line (HEK293) demonstrated that stimulation with synthetic kisspeptin-2 decapeptide (LcKiss2-10) activated multiple downstream signaling pathways, including intracellular calcium ions (Ca2+) mobilization, cyclic adenosine monophosphate (cAMP) accumulation, and mitogen-activated protein kinase /extracellular signal-regulated kinase (MAPK/ERK) phosphorylation, in a concentration-dependent manner. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis further showed that lckissr3 is expressed in multiple tissues, with the highest levels detected in the pituitary and testis. Together, these findings establish LcKissr3 as a functional kisspeptin receptor and provide a molecular basis for further investigation of kisspeptin signaling in teleost physiology.
The long-chain polyunsaturated fatty acids (LC-PUFA) are essential lipids for key physiological processes in animals. Marine polychaetes can endogenously biosynthesise these LC-PUFA through a process mediated by elongases and desaturases, making them promising candidates to support the replacement of fishmeal and fish oil in aquafeeds. Enhancing this biosynthetic ability could lead to the production of marine ingredients with improved nutritional value. This study evaluated whether dietary iron (Fe), a cofactor of fatty acyl desaturases, can enhance LC-PUFA biosynthesis in the marine polychaete Platynereis dumerilii, used here as a model species. A feeding trial was conducted under two salinity regimes (35‰ and 40‰) and two dietary treatments (Without Fe Supplementation and With Fe Supplementation), assessing growth, expression of key desaturases and elongases, and fatty acid profiles. The highest growth performance was recorded in worms reared at 40‰ and fed the Fe-supplemented diet. No change in the expression of LC-PUFA biosynthesis genes was observed across treatment conditions; however, clear effects were detected at the biochemical level. Salinity and Fe significantly influenced the fatty acid composition, with polychaetes at 40‰, especially those receiving Fe, exhibiting higher proportions of LC-PUFA, notably arachidonic acid and 20:3n-6. These results indicate that Fe can enhance LC-PUFA biosynthesis when the pathway is metabolically activated, such as under elevated salinity. Overall, this study identifies salinity and dietary Fe as complementary factors that increase LC-PUFA levels in a polychaete model, providing valuable insights for strategies aimed at enhancing the nutritional quality of marine invertebrates for sustainable aquaculture.
Chinese sturgeon (Acipenser sinensis), a critically endangered fish species, faces significant challenges in artificial propagation, with sperm quality being a key limiting factor. This study investigated the relationships among sperm quality parameters, serum reproductive hormone concentrations, and the expression of membrane-bound progesterone receptors (membrane progestin receptor α (mprα) and progesterone receptor membrane component 1 and 2 (pgrmc1 and pgrmc2)) in sexually mature male Chinese sturgeon during the spawning season. Computer-assisted sperm analysis (CASA) revealed that the normospermic group exhibited significantly higher sperm motility, A-grade sperm proportion, and mean angular displacement (MAD) than the asthenospermic group. Serum hormone assays showed that the concentrations of 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-DHP), 11-keto testosterone (11-KT), and luteinizing hormone (LH) were markedly elevated in the normospermic group. Molecular identification and bioinformatics analysis confirmed the presence of mprα, pgrmc1, and pgrmc2 in Chinese sturgeon, revealing their structural characteristics and high conservation across vertebrates. Tissue distribution analysis demonstrated sex-specific expression patterns of these genes across various tissues (pituitary, gonad, liver, heart, skin, intestine, and gill) in two-year-old fish. Notably, mRNA expression levels of these genes were significantly down-regulated in the asthenospermic group. These findings suggest that impaired progesterone signaling and endocrine dysregulation may contribute to reduced sperm motility. Our results provide foundational insights into the role of membrane-bound progesterone receptors in Chinese sturgeon reproduction, offering potential biomarkers for sperm quality assessment and targets for improving artificial breeding strategies in this endangered species.
β-carotene-15,15-dioxygenase (BCO1) and β-carotene-9,10-dioxygenase (BCO2) are essential enzymes that catalyze the oxidative cleavage of carotenoids, playing key roles in carotenoid homeostasis and pigmentation. In this study, HcBCO1 and HcBCO2 were identified from freshwater pearl mussel Hyriopsis cumingii to investigate their potential roles in carotenoid metabolism and shell nacre color formation. HcBCO1 consisted of a 1560 bp ORF, a 171 bp 5' UTR and a 171 bp 3' UTR, HcBCO2 consisted of a 1590 bp ORF, a 3 bp 5' UTR and a 394 bp 3' UTR. qRT-PCR revealed that HcBCO1 and HcBCO2 were expressed in all detected tissues, with the white shell strain showed significantly higher expression levels than purple and golden strains. By in situ hybridization, strong positive signals of HcBCO1 and HcBCO2 were detected in the outer fold (OF), middle fold (MF), inner fold (IF), and ventral mantle (VM). Further functional analysis showed that recombinant HcBCO1 and HcBCO2 expressed in E. coli were capable of cleaving β-carotene, with the pGEX-4 T-HcBCO1 group exhibiting a stronger cleavage ability. In addition, dsRNA interference demonstrated that suppressing HcBCO1/HcBCO2 expression increased carotenoid accumulation in the mantle. Our results indicate that both HcBCO1 and HcBCO2 are capable of cleaving carotenoids, with HcBCO1 exhibiting superior activity. This study offers new insights into shell color diversity in bivalves and provides potential molecular markers for genetic improvement of color and optimization of pearl hue through carotenoid metabolism regulation.
