In order to optimize the indoor scale artificial culture of Sepia pharaonis, this study compared the growth performance, composition of body components, activity of digestive enzymes, and intestinal microorganisms of S. pharaonis under different feeding frequencies, 1 time/day (T1), 2 times/day (T2), and 3 times/day (T3). The results showed that the feeding frequency had a significant effect on the growth performance, body composition, digestive enzyme activity and intestinal microorganisms of the S. pharaonis. The T3 group exhibited significantly higher survival rate, specific growth rate, weight gain rate and feed conversion ratio compared to other groups, reaching 30.76 %, 15.05 %, 31.19 % and 41.05 %, respectively. The T2 group showed the highest protein content and lowest lipid content in body composition. Digestive enzyme analysis revealed maximum amylase and protease activities in T2, while lipase activity peaked in T3. Intestinal microbiota characterization indicated a healthier and more stable microbial environment in T2. In conclusion, we suggest that the S. pharaonis (juvenile stage) cultured in the indoor cement pool environment, if the pursuit of yield, choose to feed 3 times/day, if the pursuit of quality and cost control, choose to feed 2 times/day. These findings provide crucial theoretical guidance for optimizing feeding strategies in S. pharaonis aquaculture systems.

This study investigated the effects of different nitrogen (urea, nitrate, ammonium), iron (ferric citrate, ferric chloride, ferrous sulfate), and phosphorus (monopotassium phosphate, dipotassium phosphate, calcium phytate) sources on the growth, chlorophyll a, total lipids, and fatty acid composition of the diatom Chaetoceros lorenzianus using single-factor experiments. Among nitrogen sources, ammonium and urea promoted the highest biomass, while urea also maximized chlorophyll a content. Notably, ammonium significantly enhanced the yield of polyunsaturated fatty acids (PUFA) and eicosapentaenoic acid (EPA) compared to nitrate. For iron sources, ferric chloride (FeCl3) was the most effective, improving growth, chlorophyll a, and the proportions of PUFA and EPA, without affecting total lipid content. Phosphorus sources did not influence growth significantly; however, calcium phytate and monopotassium phosphate increased total lipid yield, whereas dipotassium phosphate favored the accumulation of PUFA and EPA. This comparative analysis provides a foundational basis for selecting nutrient sources to tailor the biochemical profile of C. lorenzianus for potential applications.

Reactive oxygen species (ROS) burst represents a primary innate immune mechanism of aquatic animals to eliminate invading pathogens. Glutathione (GSH) is a key molecule in mitigating oxidative stress, yet how bacterial pathogens coordinate its synthesis and import remains unclear. This study investigated the molecular mechanism of GSH-mediated antioxidant defense in Vibrio alginolyticus H1, a pathogen isolated from the diseased cuttlefish Sepia pharaonis. The strain possesses a complete endogenous GSH synthesis cluster (gshAB) and an ABC transporter cluster (gsiABC) for exogenous GSH uptake. Under H₂O₂ stress, the mRNA expression levels of gsiA, gsiB, gsiC, gshA, and gshB increased 1.89-, 1.96-, 2.02-, 4.53-, and 5.18-fold, respectively. The growth of V. alginolyticus was inhibited in the presence of H₂O₂, but this inhibition could be alleviated by exogenous GSH supplementation. Extracellular non-enzymatic scavenging, endogenous GSH biosynthesis, and GsiABC-mediated uptake of exogenous GSH jointly enhanced the antioxidant defense of V. alginolyticus against H₂O₂-induced oxidative stress in vitro. Furthermore, a ΔgsiB mutant was constructed and showed a 6-h delay in growth recovery and impaired H₂O₂ scavenging under oxidative stress. Recombinant GsiB was shown to bind GSH directly, and key binding residues of Asp59, Lys63, Arg270, and Arg355 were identified by molecular docking. In addition, the virulence-associated phenotypes of hemolytic activity and swimming motility were attenuated in ΔgsiB. Phylogenetic analysis revealed that GsiB homologs are widely conserved across diverse bacterial species. Collectively, our findings indicate that endogenous glutathione biosynthesis and GsiB-dependent glutathione uptake jointly contribute to the antioxidant defense that may help V. alginolyticus withstand oxidative stress, as modeled by H₂O₂ challenge in vitro.

Nutrient accumulation and pathogenic Vibrio proliferation in greenhouse aquaculture effluent of Penaeus vannamei pose significant threats to sustainable shrimp production. This study evaluated the remediation efficacy of sodium alginate-immobilized Nannochloropsis oculata on real P. vannamei greenhouse aquaculture effluent under ambient greenhouse conditions. Microalgae harvested during the exponential growth phase were encapsulated via sodium alginate gelation, and the effects of algal bead dosages (0, 1, 2, and 3 g/L) on the removal of inorganic nitrogen species, phosphorus, and total Vibrio were systematically assessed. Results demonstrated dose-dependent enhancement in nutrient removal. NH₄⁺-N was removed most rapidly, with the 3 g/L group achieving 98.08 ± 1.58% removal by day 3, significantly exceeding the control (31.73 ± 2.8%; P < 0.05). PO₄³ ⁻-P removal in all treatment groups surpassed 96% by day 3. NO₃⁻-N and NO₂⁻-N removal exhibited a temporal lag consistent with the sequential nitrogen assimilation hierarchy (NH₄⁺-N > NO₃⁻-N > NO₂⁻-N), reaching 95.44 ± 0.91% and 85.71 ± 1.24% in the 3 g/L group by day 5 (P < 0.05). TIN and TIP removal rates in the 3 g/L group reached 88.70 ± 1.37% and 83.49 ± 2.25%, representing 3.48- and 4.57-fold improvements over the control, respectively. Additionally, total Vibrio counts in the 3 g/L group decreased to 11.42 ± 0.68 CFU/mL by day 5, compared with 122.20 ± 5.36 CFU/mL in the control (P < 0.05). Reusability assessments indicated that the system retained high efficiency across two treatment cycles, though bead structural degradation in Cycle 3 compromised performance, suggesting a two-cycle reuse protocol. These findings demonstrate that immobilized N. oculata at 3 g/L achieves optimal effluent purification with dual functionality in nutrient bioremediation and pathogenic bacterial suppression, providing a scientific basis for scalable implementation in greenhouse aquaculture wastewater management.

Sodium hypochlorite (NaClO) is widely used in aquaculture disinfection, however, its safety thresholds for sensitive cephalopod species remain undefined. This study systematically evaluated the toxicological effects of NaClO on embryonic development, growth performance, and physiological homeostasis in the Pharaoh cuttlefish (Sepia pharaonis). Fertilized eggs were exposed to NaClO at gradient concentrations of 1.0-7.0 mg/L for durations of 1, 5, and 10 min, while juveniles were exposed at concentrations of 1.0-4.0 mg/L for durations of 1 and 3 min. The results revealed a pronounced dose-and time-dependent toxicity profile. Mild exposure (1 mg/L for 1 min) maintained high hatching rates (93.3%) comparable to controls (92.2%), extreme exposure (7 mg/L for 10 min) drastically reduced hatching success to 25.6% and impaired hatchling morphology (P < 0.05). In juveniles, survival and specific growth rates were markedly suppressed at concentrations >= 3 mg/L compared to the controls. Physiological analysis revealed a stress-induced metabolic shift: muscle glycogen content and glycolytic enzyme activities (hexokinase and pyruvate kinase) increased dose-dependently, indicating a compensatory transition to anaerobic metabolism. Although antioxidant enzymes (SOD, CAT, GSH-Px) and HSP70 gene expression were upregulated to counteract oxidative stress, these defense mechanisms were overwhelmed at higher concentrations (3-4 mg/L), culminating in substantial lipid peroxidation (elevated MDA) and severe histopathological degeneration in the digestive gland, characterized by cellular lysis and apoptosis. Principal Component Analysis (PCA) confirmed that NaClO concentration was the predominant determinant of physiological alteration. Consequently, this study establishes a critical safety threshold of <= 2 mg/L for <= 1 min for S. pharaonis, providing evidence-based guidelines for optimizing disinfection protocols in industrial-scale cephalopod hatchery operations.

The marine planktonic diatom genus Corethron has predominantly been studied for its growth responses to environmental factors. However, research on the effects of nutrient salts on its growth, total lipid content, and fatty acid composition, as well as scalable cultivation and comprehensive utilization, remains limited. This study investigated the impacts of nitrogen, phosphorus, iron, and silicon forms and concentrations on the growth and lipid biosynthesis of Corethron sp. Single-factor experiments established the optimal culture conditions as follows: Nitrogen source is NaNO2 at a concentration of 20 g L⁻1, phosphorus source is KH2PO4 at a concentration of 1 g L⁻1, iron source is FeC6H5O7 at a concentration of 0.1 g L⁻1, and silicon source is Na2SiO3 at a concentration of 10 g L⁻1. Comparative experiments using two nutrient salt formulations showed that the modified stock solution significantly enhanced total lipid content by 16.6

This study aimed to assess the effects of Isochrysis galbana, Streptotheca thamesis, and Chaetoceros gracilis as diets in the rearing of juvenile Ruditapes philippinarum. We evaluated the effects of these microalgae and their mixed diet on the feeding efficiency, growth, nutritional metabolism, and digestive enzyme activity of juvenile R. philippinarum through feeding trials, growth performance analysis, and biochemical assays. Different microalgal diets significantly influenced the feeding efficiency of juvenile clams. The highest filtration rate was observed in the C. gracilis group (p < 0.05), and all groups had survival rates above 97

In order to optimize the culture conditions of the diatom Chaetoceros lorenzianus, effects of phosphorus sources (K2HPO4, KH2PO4, CaP2H4O8) and phosphorus concentrations (0.50, 1.00, 1.50, 2.00, 2.50, 3.00 mg L−1) on C. lorenzianus were studied. The results showed that phosphorus sources and phosphorus concentrations had significant effects on the growth, chlorophyll a, total lipid and fatty acids of C. lorenzianus. The growth rate, chlorophyll a and total lipids production, and MUFA (monounsaturated fatty acid) composition were significantly higher in the CaP2H4O8 group when compared to K2HPO4 and KH2PO4 groups. The optimal concentration of CaP2H4O8 for growth, chlorophyll a and total lipid accumulation was 1.00 mg L−1, and the optimum concentration for PUFA (Polyunsaturated fatty acid), EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid) were 2.00 mg L−1. The results of this study provide important theoretical guidance for the future development and utilization of C. lorenzianus, including algal culture, optimization of fatty acid composition, and have great potential economic value in the field of feed and food development.

In order to investigate the causes of population degradation and resource decline, this thesis investigated the ecotoxicological effects of heavy metal Cu(II) on the embryonic development of Sepiella maindroni. Results indicate significant effects of Cu(II) concentrations on the developmental toxicity, teratogenicity, and lethality of S. maindroni embryos. Different concentrations of Cu(II) caused varying degrees of malformations in embryos, altered developmental rates, reduced hatchability and hatchling quality, and increased malformation and mortality of hatchlings. At the same time, Cu(II) exposure led to an increase in the content of the lipid peroxidation product malondialdehyde (MDA) and a significant decrease in the activity of antioxidant enzymes (superoxide dismutase [SOD], catalase [CAT]), energy-metabolizing enzymes (adenylate kinase [AK]), and cholinergic-related enzymes (acetylcholinesterase [AChE], choline acetyltransferase [ChAT]). In conclusion, when the concentration of Cu(II) in the environment is >= 0.01 mg/L, it causes significant lethality toxicity, developmental toxicity and teratogenicity in S. maindroni embryos. These effects are likely related to Cu(II)induced stress impacting the antioxidant capacity, energy metabolism, and cholinergic system. Ultimately, these toxic effects may lead to population degradation and resource decline in fishery organisms by affecting the early replenishment process of fisheries.

Ink-jetting is a key defense mechanism in cephalopods, but excessive discharge can cause metabolic imbalance and oxidative tissue damage. While previous studies have focused on tissue-level responses, systemic metabolic adaptation and amino acid redistribution remain poorly understood. Here, Sepia pharaonis was subjected to repeated ink-jetting stress, and hematological parameters and free amino acids (FAAs) concentrations in muscle and the ink sac were analyzed over a 28-day recovery period. Ink-jetting triggered rapid metabolic mobilization, with blood glucose increasing from 0.50 ± 0.06 mmol/L to 0.92 ± 0.05 mmol/L (+ 84.13

Tributyltin (TBT) is known for its environmental persistence and high toxicity, posing a significant threat to benthic aquatic organisms in coastal zones. The present study employed physiological, histological, and multiomics techniques to investigate the toxic effects of TBT exposure and the detoxification mechanisms in Sepia pharaonis. The results revealed that TBT exposure resulted in reduced growth performance, elevated activity of the antioxidant enzyme system, and pronounced histopathological alterations in the digestive glands, suggesting substantial oxidative stress within these tissues. Transcriptome analysis indicated that differentially expressed genes were significantly enriched in pathways related to reactive oxygen species (ROS) metabolism, oxidative stress, the mitochondrial respiratory chain, antioxidant activity, and stress responses. Furthermore, levels of metabolites involved in ROS scavenging-including oxidized glutathione, L-arginine, L-glutamate, gamma-glutamyl-Lalanine, and L-glycine-were markedly elevated, reflecting the organism's response to reduce the excess ROS induced by TBT stress. Additionally, the integrated analysis of transcriptome and metabolome data indicated that the cuttlefish could effectively counteract TBT-induced oxidative stress via its antioxidant enzyme system. However, exposure to high concentrations of TBT prompted a shift from reliance on the antioxidant enzyme system to the activation of detoxification defense mechanisms, with a pronounced effect on glutathione metabolism and arginine biosynthesis. In conclusion, our findings enhance the understanding of S. pharaonis's adaptability to TBT-stressed environments and offer new insights into the molecular mechanisms underlying TBT-induced detoxification.

Vibrio alginolyticus is a marine opportunistic bacterium that can infect a variety of aquatic animals. In this study, V. alginolyticus H1 was found to degrade collagen from the skin of the cuttlefish Sepia pharaonis, therefore; a colA gene encoding collagenase was cloned from V. alginolyticus H1. The expression at the mRNA level and enzymatic activity of ColA under different redox pressures were determined using real time RT-PCR and gelatin biochemical tubes. The catalytic domain of ColA was recombined in Escherichia coli BL21(DE3), recombinant ColA-CD (rColA-CD) was purified via Ni-NTA agarose, and the optimum temperature and pH for rColA-CD were approximately 40 degrees C and 9.0, respectively. To further investigate the role of ColA in bacterial virulence, the colA gene was knocked out to construct the Delta colA mutant, which exhibited reduced virulence to S. pharaonis compared with that of the wild type (WT) strain. Transcriptomic analysis revealed that, compared with the cuttlefish S. pharaonis infected with WT, the individuals infected with Delta colA showed 392 differentially expressed genes (DEGs), including 111 upregulated DEGs and 281 downregulated DEGs, among which the level of mammalian matrix metalloproteinase 19 was downregulated. Immune-related pathways, such as the ErbB signaling pathway, and JAK-STAT signaling pathway were downregulated, whereas protein processing in the endoplasmic reticulum was upregulated. This study demonstrated that the collagenase encoded by the colA gene is a critical virulence factor of V. alginolyticus, which regulates specific immune-related pathways in the cuttlefish S. pharaonis.

The goals of this research were to resolve the problems of low quantity and poor quality of eggs in cuttlefish aquaculture, and to provide empirical support for the development of formulated feeds. The impacts of different feeds on the quantity and quality of eggs laid by cuttlefish ( Sepia pharaonis ) were studied using three different species: Litopenaeus vannamei, Nibea albiflora , and Sillago sihama . We explored the influence of dietary composition on the quantity and quality of eggs laid. The results showed that the quantity and quality of eggs laid were significantly affected by the feed species. Compared with the feed of S. sihama and N. albiflora , the feed of L. vannamei increased the number of eggs laid by17.45% and 24.15%, the egg width by 5.05% and 5.91%, the hatching rate by 5.42% and 12.59%, and the weight of newly hatched cuttlefish by 5.05% and 5.98%, and there was no significant difference in the incubation period. The quantity and the quality (the egg width, incubation period, hatching rate, and weight of newly hatched cuttlefish) were significantly positively correlated with protein content, C18:1(n‐9), C18:2, C18:3, C20:2, C20:3, arachidonic acids (AAs), eicosapentaenoic acids (EPAs), and docosahexaenoic acid (DHA) of feed. These results demonstrate that L. vannamei is the optimal feed for broodstock cuttlefish during the breeding period, and that diets rich in protein and n‐3 highly unsaturated fatty acids (HUFA) can enhance their reproductive performance.

The objective of this study was to identify the most suitable tank colors for rearing juvenile pharaoh cuttlefish. Six tank colors (red, yellow, white, green, blue, black) were evaluated to determine their effects on the growth performance, body color, stress response, energy metabolism (hexokinase (HK), pyruvate kinase (PK), arginine kinase (AK)), and antioxidant capacity (superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA)) of juvenile cuttlefish. The results indicate that tank color had a significant impact on various parameters including specific growth rate, weight gain rate, survival rate, feed coefficient, stress response, energy metabolism, and antioxidant capacity in juvenile cuttlefish (P < 0.05). Regarding growth performance, juvenile cuttlefish in the red and blue groups demonstrated the highest specific growth rate, weight gain rate, and survival rate, along with the lowest feed coefficient (P < 0.05), indicating optimal growth conditions, whereas the green and white groups showed contrasting results (P < 0.05). Juvenile cuttlefish in the red and black groups exhibited a light gray body color, whereas those in the blue group displayed a brown coloration; individuals in the white, yellow, and green groups exhibited a dark brown coloration. Concerning stress response, cortisol levels were highest in the yellow and green groups (P < 0.05), indicating the highest stress levels, whereas they were lowest in the red and black groups (P < 0.05). Regarding energy metabolism, HK, PK, and AK levels were highest in the yellow and green groups (P < 0.05), suggesting greater energy demands, whereas they were lowest in the red and black groups (P < 0.05). As for antioxidant capacity, the white, yellow, and green groups demonstrated the highest SOD and CAT activities (P < 0.05), while the green and yellow groups additionally exhibited the highest MDA content (P < 0.05), suggesting elevated antioxidant capacity levels; conversely, the red and black groups displayed the lowest CAT and SOD activities and MDA content (P < 0.05). In conclusion, red tanks are advantageous in enhancing the specific growth rate, weight gain rate, survival rate, and feed coefficient of juvenile cuttlefish, while also mitigating stress responses and maintaining metabolic stability, rendering them more suitable for the cultivation of juvenile cuttlefish.

Thalassiosira pseudonana, a fast-growing and nutritionally rich planktonic diatom, has been cultivated on a large scale as high-quality plankton feed. Despite this, studies exploring the impact of light quality on the growth and compositional attributes of this algal species are scarce. This study investigated the effects of five different light qualities—white, red, yellow, green, and blue—on the growth, biomass, fucoxanthin accumulation, and fatty acid composition and content of T. pseudonana. The results indicated that T. pseudonana achieved the highest growth rate and greatest biomass under white and blue light, with the poorest performance observed under red light. Moreover, fucoxanthin accumulation was most pronounced under blue light, reaching 4.729 ± 0.182 mg g-1, followed in descending order by yellow, red, white, and green light. The total lipid content was highest under yellow light at 34

To investigate the impact of different light qualities (red, blue, green, yellow, and white light) on the embryonic development of Sepia pharaonis, this experiment studied the effects of different light qualities on the timing of embryonic development, incubation period, hatching rate, malformation rate, weight of hatchlings, and survival rate on the 7th day after hatching. The findings demonstrated a significant influence of different light qualities on the embryonic development of S. pharaonis (P<0.05). Specifically, red and blue light led to increased occurrences of phenomena such as yolk surface cracking, cell autolysis, coagulation of cells in the animal pole, yolk sac edema, malformations in endoskeleton development, and decreased pigmentation and whitening of the body surface compared to the control group (white light). Conversely, green light showed a tendency to reduce these phenomena. The hatching rate and hatching cuttlefish weight were highest under green and yellow light conditions, with significantly greater survival rate on the 7th day after hatching compared to red, blue, and white light conditions. Furthermore, the malformation rate was notably lower under green light conditions compared to other light groups. Red and blue light conditions exhibited the shortest hatching periods, significantly outpacing other light groups. In conclusion, green light appears advantageous for enhancing hatching rate, the quality and health of hatched larvae, reducing malformation occurrences and embryonic mortality, and improving overall hatching quality.

Skin ulcer disease causes massive mortalities during the culture of the cuttlefish Sepia pharaonis. In the present study, metabolomic and 16S rRNA sequence were used to investigate the variation in metabolites and bacterial communities in the skin tissue of S. pharaonis with skin ulcer symptom. S. pharaonis were collected at the undiseased stage (UDS), early diseased stage (EDS), and agonal stage (AS) during a severe and highly contagious skin ulcer disease on the fin. Significantly lysis of the epidermal cells in the skin tissue at both the AS and EDS groups were observed under microscope. Specific metabolic changes responding to disease were identified using metabolomic analysis. Choline continually increased while osmotic chemicals including betaine, homotaurine, and trehalose, decreased; variable levels of phosphocholine and AMP related to energy, and decreased GSH/GSSG ratio related to intense oxidative stress disturbed the balance of osmoregulators, energy and redox equilibrium. 16S rRNA sequence showed a significant decrease in Pseudomonas spp. and Acinetobacter spp., but an increase in Vibrio spp. as the disease progressed. Gammaproteobacteria and Bacillus spp. can be used as biomarkers for the skin ulcer disease, while Pseudomonas spp. and Acinetobacter spp. were the potential probiotics for S. pharaonis culturing. Our present results indicated the characteristic changes in metabolic profile and bacterial community as the disease progressed, which would offer basic information for the further understanding of the disease occurrence in S. pharaonis culturing.

Research on the aquaculture of cephalopods has emerged in the last 30 years. Compared to other animals, limited information exists on the fundamental biology of cephalopods, particularly regarding their gonadal and embryonic development. Detailed investigation into their reproduction and development could provide a solid practical basis for understanding their methods of reproduction and reproductive patterns for enhancing artificial-seeding cultivation techniques. Therefore, this study utilised the second generation (F2) of cultured cuttlefish, Sepia pharaonis Ehrenberg, 1831, controlling the hatching temperature of fertilised eggs and recording data at various stages of gonadal development. We calculated the biological zero, the effective cumulative temperature, the temperature coefficient for embryonic development and the effective cumulative temperature for gonadal development. The results indicated that the biological zero and effective cumulative temperature for embryonic development of S. pharaonis were 7.46 and 434.87 degrees C.d, respectively, with the optimal temperature range for embryonic development being 24-27 degrees C. The effective cumulative temperature for gonadal development was 853.04 +/- 57.51 degrees C.d. This study provides essential information on the biological zeros and effective cumulative temperatures for both embryonic and gonadal development of S. pharaonis, offering significant practical information and a theoretical basis for future large-scale artificial cultivation of this species.