Biochemical indicators, cell apoptosis, and metabolomic analyses of the low-temperature stress response and cold tolerance mechanisms in Litopenaeus vannamei.

The cold tolerance of Litopenaeus vannamei is important for breeding in specific areas. To explore the cold tolerance mechanism of L. vannamei, this study analyzed biochemical indicators, cell apoptosis, and metabolomic responses in cold-tolerant (Lv-T) and common (Lv-C) L. vannamei under low-temperature stress (18 °C and 10 °C). TUNEL analysis showed a significant increase in apoptosis of hepatopancreatic duct cells in L. vannamei under low-temperature stress. Biochemical analysis showed that Lv-T had significantly increased levels of superoxide dismutase (SOD) and triglycerides (TG), while alanine aminotransferase (ALT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH-L), and uric acid (UA) levels were significantly decreased compared to Lv-C (p < 0.05). Metabolomic analysis displayed significant increases in metabolites such as LysoPC (P-16:0), 11beta-Hydroxy-3,20-dioxopregn-4-en-21-oic acid, and Pirbuterol, while metabolites such as 4-Hydroxystachydrine, Oxolan-3-one, and 3-Methyldioxyindole were significantly decreased in Lv-T compared to Lv-C. The differentially regulated metabolites were mainly enriched in pathways such as Protein digestion and absorption, Central carbon metabolism in cancer and ABC transporters. Our study indicate that low temperature induces damage to the hepatopancreatic duct of shrimp, thereby affecting its metabolic function. The cold resistance mechanism of Lv-T L. vannamei may be due to the enhancement of antioxidant enzymes and lipid metabolism.

Transcriptomic analysis reveals nanoplastics-induced apoptosis, autophagy and immune response in Litopenaeus vannamei.

Increasing attention is being paid to the toxic physiological effects of nanoplastics (NPs) on aquatic organisms. However, few studies have systematically evaluated the regulatory mechanisms of NPs on immune response in crustaceans. In this study, a 28-day chronic exposure experiment was conducted in which shrimps were exposed to various 80-nm polystyrene NPs concentrations (0, 0.1, 1, 5 and 10 mg/L). Transcriptomic analysis was used to investigate the regulatory mechanisms of NPs in immune response of Litopenaeus vannamei. With increasing NPs concentration, the total hemocyte count (THC) content decreased, while phagocytosis rate (PR) and respiratory burst (RB) showed trends of first rising and then falling. High concentration (10 mg/L) of NPs caused the destruction of hepatopancreas tissue structure, the shedding of microvilli, the increase number of hepatocyte apoptosis and autophagy structure. With increasing NPs concentration, the lysozyme (Lys), superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities first increased and then decrease, while contents of lipid peroxidation and malondialdehyde increased; the expression levels of Toll, MyD88, GPx, SOD, proPO, Lys, and ALF generally increased at first and then decreased. Transcriptional sequencing analysis showed that the pathway of differentially expressed genes in KEGG enrichment mainly included lysosome (ko04142), apoptosis (ko04210) pathways, indicating that the NPs mainly affected the immune regulatory mechanism. Further analysis by Gene Set Enrichment Analysis (GSEA) showed that the up-regulation pathways of NPs activation mainly included immune response-related pathways such as mitochondrial autophagy, DNA repair, autophagosomes signaling pathway. Our results indicated that NPs exposure induced oxidative stress, apoptosis and autophagy in shrimps. This study provides a basis for further understanding of the mechanisms of antioxidant immune regulation by NPs in shrimp and may serve as a reference for healthy ecological culture of shrimp.

Automatic Shrimp Fry Counting Method Using Multi-Scale Attention Fusion.

Shrimp fry counting is an important task for biomass estimation in aquaculture. Accurate counting of the number of shrimp fry in tanks can not only assess the production of mature shrimp but also assess the density of shrimp fry in the tanks, which is very helpful for the subsequent growth status, transportation management, and yield assessment. However, traditional manual counting methods are often inefficient and prone to counting errors; a more efficient and accurate method for shrimp fry counting is urgently needed. In this paper, we first collected and labeled the images of shrimp fry in breeding tanks according to the constructed experimental environment and generated corresponding density maps using the Gaussian kernel function. Then, we proposed a multi-scale attention fusion-based shrimp fry counting network called the SFCNet. Experiments showed that our proposed SFCNet model reached the optimal performance in terms of shrimp fry counting compared to CNN-based baseline counting models, with MAEs and RMSEs of 3.96 and 4.682, respectively. This approach was able to effectively calculate the number of shrimp fry and provided a better solution for accurately calculating the number of shrimp fry.

Influence of stocking density on the growth, immune and physiological responses, and cultivation environment of white-leg shrimp (Litopenaeus vannamei) in biofloc systems.

Biofloc (BF) stands out as a promising system for sustainable shrimp farming. Optimizing various culture conditions, such as stocking density, carbohydrate source, and feeding management, is crucial for the widespread adoption of the BF system. This study compares the growth performance of white-leg shrimp (Litopenaeus vannamei) in culture ponds at low density (LD) with 50 organisms/m2 and high density (HD) with 200 organisms/m2. Post-larvae of white-leg shrimp were stocked for 16 weeks in both LD and HD groups. The LD group exhibited a superior survival rate, growth rate, and feed consumption compared to the HD group. The BF from the LD system recorded a significantly higher protein content (16.63 ± 0.21%) than the HD group (15.21 ± 0.34%). Heterotrophic bacterial counts in water did not significantly differ with stocking density. However, Vibrio count in water samples was higher in the HD group (3.59 ± 0.35 log CFU/mL) compared to the LD group (2.45 ± 0.43 log CFU/mL). The whole shrimp body analysis revealed significantly higher protein and lipid content in the LD group. In contrast, the total aerobic bacterial count in shrimp from the HD group was high, with the identification of Salmonella enterica ssp. arizonae. Additionally, Vibrio counts in shrimp samples were significantly higher in the HD group (4.63 ± 0.32 log CFU/g) compared to the LD group (3.57 ± 0.22 log CFU/g). The expression levels of immune-associated genes, including prophenoloxidase, transglutaminase, penaiedin 3, superoxide dismutase, lysozyme, serine proteinase, and the growth-related gene ras-related protein (rap-2a), were significantly enhanced in the LD group. Conversely, stress-related gene expression increased significantly in the HD group. Hepatopancreases amylase, lipase, and protease were higher in the LD group, while trypsin activity did not differ significantly. Antioxidant enzyme activity (catalase, glutathione, glutathione peroxidase, and superoxide dismutase) significantly increased in the LD group. The histological structure of hepatopancreas, musculature, and female gonads remained similar in both densities. However, negative effects were observed in the gills' histology of the HD group. These results suggest that increasing stocking density is associated with significantly negative biological, microbial, and physiological effects on white-leg shrimp under the BF system.

Dietary lipid supplementation alleviated the impacts of polystyrene nanoplastic exposure in Litopenaeus vannamei.

The widespread occurrence of nanoplastic (NP) pollution in the environment is a growing concern, and its presence poses a potential threat to cultured aquatic animals. Previously, we found that NPs can significantly affect the lipid metabolism of shrimp. However, relevant reports about the effects of increasing dietary lipid levels on NP toxicity are lacking. Therefore, we explored the effects of dietary supplementation with different lipid levels on the growth and lipid metabolism of Pacific white shrimp (Litopenaeus vannamei). We cultured L. vannamei at three dietary lipid levels (3 %, 6 %, and 9 %) and three NP concentrations (0, 1, and 3 mg/L) for 2 months. We evaluated the effects of lipid levels on growth indexes, hepatopancreas morphological structure, lipid metabolism-related enzyme activity, and gene expression of the shrimp. The results showed that as lipid intake increased, the survival rate, body weight growth rate, and hepatosomatic ratio of the shrimp increased while the feed conversion rate decreased. Additionally, the crude protein and crude lipid contents increased, whereas the moisture and ash contents did not change much. We found that the morphological structure of the hepatopancreas was seriously damaged in the 3 mg/L NPs and 3 % dietary lipid group. Finally, lipid metabolism-related enzyme activities and gene expression levels increased with increased dietary lipid levels. Together, these results suggest that increasing dietary lipid content can improve shrimp growth and alleviate lipid metabolism disorders caused by NPs. This study is the first to show that nutrition regulation can alleviate the toxicity of NPs, and it provides a theoretical basis for the green and healthy culture of L. vannamei.

Effect of temporal thermal stress on Penaeus vannamei: Growth performance and physiological plasticity.

The present study evaluated the effect of temporal periods of hypothermia and hyperthermia, followed by an optimal temperature recovery phase on the growth, survival, and physiological response of Penaeus vannamei. Post-larvae were exposed to stress periods for 7 and 14 days at 22 °C and 32 °C each, followed by a recovery phase at 28 °C to complete seven experimental weeks, and were compared with a control group maintained at 28 °C. Weight gain, specific growth rate, feed intake, feed conversion ratio, and survival were weekly determined. Muscle, hepatopancreas, and hemolymph were sampled on the 14th day of the recovery phase for biochemical composition, and antioxidant and digestive enzyme activities determination. The shrimp presented a higher growth rate during short-term hyperthermia in contrast to shrimp under hypothermia that presented compensatory growth after thermal stress when the temperature was restored at 28 °C. Hyperthermia increased 12-13% the feed intake while this was diminished 21-29% by the hypothermia periods. Shrimp undergo metabolic adjustments following thermal stress, with short hypothermia increasing the lipase activity and lipid storage in the hepatopancreas, while short hyperthermia also enhances chymotrypsin activity and leads to higher protein and lipid accumulation. Conversely, prolonged hyperthermia induces greater energy consumption, depleting lipid and glycogen stores, while hypothermia causes scarce mobilization of energy reserves during recovery phase. Antioxidant enzyme activities were not affected by short-thermal stress (7d), while prolonged thermal stress (14d) significantly affected SOD, CAT, and GPx activities. The present study provides important insights into the physiological plasticity of P. vannamei during recovery from thermal stress.

Protective effects of butyric acid during heat stress on the survival, immune response, histopathology, and gene expression in the hepatopancreas of juvenile pacific shrimp (L. Vannamei).

This study looked at the effects of adding butyric acid (BA) to the diets of juvenile Pacific shrimp and how it affected their response to survival, immunity, histopathological, and gene expression profiles under heat stress. The shrimp were divided into groups: a control group with no BA supplementation and groups with BA inclusion levels of 0.5 %, 1 %, 1.5 %, 2 %, and 2.5 %. Following the 8-week feeding trial period, the shrimp endured a heat stress test lasting 1 h at a temperature of 38 °C. The results showed that the control group had a lower survival rate than those given BA. Interestingly, no mortality was observed in the group receiving 1.5 % BA supplementation. Heat stress had a negative impact on the activities of alkaline phosphatase (AKP) and acid phosphatase (ACP) in the control group. Still, these activities were increased in shrimp fed the BA diet. Similar variations were observed in AST and ALT fluctuations among the different groups. The levels of triglycerides (TG) and cholesterol (CHO) increased with high temperatures but were reduced in shrimp-supplemented BA. The activity of an antioxidant enzyme superoxide dismutase (SOD) increased with higher BA levels (P < 0.05). Moreover, the groups supplemented with 1.5 % BA exhibited a significant reduction in malondialdehyde (MDA) content (P < 0.05), suggesting the potential antioxidant properties of BA. The histology of the shrimp's hepatopancreas showed improvements in the groups given BA. Conversely, the BA significantly down-regulated the HSPs and up-regulated MnSOD transcript level in response to heat stress. The measured parameters determine the essential dietary requirement of BA for shrimp. Based on the results, the optimal level of BA for survival, antioxidant function, and immunity for shrimp under heat stress is 1.5 %.

Effects of the putative probiotics Bacillus licheniformis, Bacillus pumilus, and Bacillus subtilis on white leg shrimp, Litopenaeus vannamei, immune response, gut histology, water quality, and growth performance.

Background: A commercially significant species in the aquaculture sector globally, particularly in Egypt, is Litopenaeus vannamei. Aim: The experiment's objective was to ascertain how Sanolife PRO-F impacted the growth, water quality, immunological response, and intestinal morphometry of L. vannamei. Methods: In the current investigation, which lasted 12 weeks, Sanolife PRO-F was administered to shrimp post-larvae at diet doses of 0 (control), 1 (group one), 2 (group two), and 3 (group three) g/kg diet, respectively. Each experimental group had three repetitions. Results: In the current study, shrimp fed on probiotic-treated diets showed a considerable improvement in growth performance measures and survival rate, and the nonspecific immune response was also enhanced. Shrimp fed probiotic diets had longer and more intestinal villi overall. Shrimp fed on the G2 and G3 diets showed no appreciable differences in growth or intestinal morphology. With the G2 and G3 diet, the water had lower concentrations of nitrite and ammonia. Conclusion: The study's findings indicate that Sanolife PRO-F treatment at 2-3 g/kg feed promotes the growth of shrimp, immunological response, gut health and function, and water quality.

Microfibers from cigarette butts can induce exoskeletal alteration in whiteleg shrimp (Penaeus vannamei).

Cigarette butts (CB) are a source of microfibers (MFs) in aquatic environments, posing a risk to the health of aquatic organisms. Research has been focused on examining the toxicity of CBs on ecological receptors, including invertebrates. More focus has been on death, growth, or movement inhibition of but less on exoskeletal effects in malacostracans. We evaluated the alteration in the carapace structure of whiteleg shrimp (Penaeus vannamei Boone, 1931) caused by MFs derived from CBs (CB-MF). Exposure to CB-MF damaged the gills, the main organs adsorbing calcium in shrimps to generate a hard carapace, disturbing calcium uptake via respiration. Rapid ecdysis caused on CB-MF exposure reduced the environmental adaptation capacity of crustaceans in the absence of normal pigments in the chromatophore of the carapace. These findings indicate that MFs released from CBs released into the aquatic environment can adversely affect exoskeletal alteration within the overall ecosystem.

Transcriptomic analysis following polystyrene nanoplastic stress in the Pacific white shrimp, Litopenaeus vannamei.

Plastics are widely produced for industrial and domestic applications due to their unique properties, and studies on the toxic effects of nanoplastics (NPs) on aquatic animals are essential. In this study, we investigated the transcriptomic patterns of Litopenaeus vannamei after NPs exposure. We found that the lysosome pathway was activated when after NPs exposure, with up-regulated DEGs, including glucocerebrosidase (GBA), hexosaminidase A (HEXA), sphingomyelin phosphodiesterase-1 (SMPD1), and solute carrier family 17 member 5 (SLC17A5). In addition, the PI3K-Akt signaling pathway was strongly affected by NPs, and the upstream genes of PI3K-Akt, including epidermal growth factor receptor (EGFR), integrin subunit beta 1 (ITGB1) and heat shock protein 90 (HSP90) were up-regulation. Other genes involved in lipogenesis, such as sterol regulatory element binding transcription factor 1 (SREBP-1c), fatty acid synthase (FASN) and stearoyl-CoA desaturase (SCD-1), were down-regulated. However, the contents of triglycerides (TG) and total cholesterol (TCH) in L. vanname hepatopancreas were reduced, which indicated that the ingestion of NPs led to the disturbance of hepatic lipid metabolism. What more, NPs treatment of L. vannamei also caused oxidative stress. In addition, NPs can damage part of the tissue structure and affect the physiological function of shrimps. The results of this study provide valuable ecotoxicological data to improve the understanding of the biological fate and effects of nanoplastics in L. vannamei.

Effects of Lactobacillus plantarum Ep-M17 on growth, immunity and intestinal microbiota of Penaeus vannamei under Microcystin-LR stress.

Microcystins (MCs) are biologically active cyclic heptapeptide compounds released by cyanobacteria in water bodies, and MC-LR is one of the most widespread and toxic isoforms. It frequently poses a serious threat to Penaeus vannamei aquaculture. Our previous study revealed that the supplementation of Lactobacillus plantarum Ep-M17 has a probiotic effect on P. vannamei health and whether Ep-M17 can alleviate the stressful effects of MC-LR on shrimp remains unclear. Therefore, in the present work, shrimp were fed MC-LR alone or combined with Ep-M17 for six weeks, and then evaluated the effects on histology, enzyme activity, gene expression, and intestinal flora. The results showed that MC-LR stress lead to slow growth and reduced survival rates in shrimp. However, feeding Ep-M17 significantly increased both the growth rate and survival rate. Meanwhile, MC-LR stress caused severe tissue damage in the hepatopancreas and intestines of shrimp, but Ep-M17 significantly reduced the toxic effects and protected the integrity of these tissues. Additionally, Ep-M17 significantly enhanced the activities of antioxidant enzymes and digestive enzymes, and induced higher expression of immune-related genes, thereby promoting the digestive and immune responses in shrimp. Furthermore, MC-LR stress disrupted the intestinal flora in shrimp intestines, while the use of Ep-M17 significantly increased the abundance of immune- and metabolism-related bacteria and inhibited the growth of pathogenic bacteria to maintain intestinal flora balance and intestinal health. In conclusion, our results indicate that Ep-M17 can reduce the toxic effect of MC-LR on shrimp and has a positive function in the prevention and control of shrimp diseases caused by MC-LR.

New insights into the regulation mechanism of Pacific white shrimp (Litopenaeus vannamei) hepatopancreas under 4-nonylphenol exposure using transcriptome analysis.

4-Nonylphenol (4-NP) is one of the common endocrine-disrupting chemicals (EDCs) in estuaries and coastal zones, which can exert detrimental effects on the physiological function of aquatic organisms. However, the molecular response triggered by 4-NP remains largely unknown in Pacific white shrimp (Litopenaeus vannamei). In this study, transcriptomic analysis was performed to investigate the underlying mechanisms of 4-NP toxicity in the hepatopancreas of L. vannamei. Nine RNA-Seq libraries were generated from L. vannamei at 0 h, 24 h, and 48 h following exposure to 4-NP. Compared with 0 h vs 24 h, 962 up- and 463 down-regulated differentially expressed genes (DEGs) were identified, indicating that many genes in L. vannamei were induced to resist adverse circumstances by 4-NP exposure. In contrast, 902 up- and 1027 down-regulated DEGs were revealed in the comparison of 0 h vs 48 h, demonstrating that prolonged exposure to the stress from 4-NP resulted in more inhibited genes. To validate the accuracy of the transcriptome data, eight DEGs were selected for quantitative real-time polymerase chain reaction (qRT-PCR), which were consistent with the RNA-Seq results. Through KEGG pathway enrichment analysis, three specific pathways related to hormonal effects and endocrine function of L. vannamei were enriched significantly, including tyrosine metabolism, insect hormone biosynthesis, and melanogenesis. After 4-NP stress, genes involved in tyrosine metabolism (Tyr) and melanogenesis pathway (AC, CBP, Wnt, Frizzled, Tcf, and Ras) were induced to promote melanin pigment to help shrimp resist adverse environments. In the insect hormone biosynthesis, ALDH, CYP15A1, CYP15A1/C1, and JHE genes were activated to synthesize juvenile hormone (JH), while Spook, Phm, Sad, and CYP18A1 were induced to generate molting hormone. There is an enhanced interaction between the molting hormone and JH, with JH playing a dominant role and maintaining its "classic status quo action". Our study demonstrated that 4-NP exposure led to impairments of biological functions in L. vannamei hepatopancreas. The genes and pathways identified provide novel insights into the molecular mechanisms underlying 4-NP toxicity effects in prawns and enrich the information on the toxicity mechanism of crustaceans in response to EDCs exposure.

Penaeid shrimp counteract high ammonia stress by generating and using functional peptides from hemocyanin, such as HMCs27.

Agricultural and anthropogenic activities release high ammonia levels into aquatic ecosystems, severely affecting aquatic organisms. Penaeid shrimp can survive high ammonia stress conditions, but the underlying molecular mechanisms are unknown. Here, total hemocyanin and oxyhemocyanin levels decreased in Penaeus vannamei plasma under high ammonia stress. When shrimp were subjected to high ammonia stress for 12 h, 24 hemocyanin (HMC) derived peptides were identified in shrimp plasma, among which one peptide, designated as HMCs27, was chosen for further analysis. Shrimp survival was significantly enhanced after treatment with the recombinant protein of HMCs27 (rHMCs27), followed by high ammonia stress. Transcriptome analysis of shrimp hepatopancreas after treatment with or without rHMCs27 followed by high ammonia stress revealed 973 significantly dysregulated genes, notable among which were genes involved in oxidation and metabolism, such as cytochrome C, catalase (CAT), isocitrate dehydrogenase, superoxide dismutase (SOD), trypsin, chymotrypsin, glutathione peroxidase, glutathione s-transferase (GST), and alanine aminotransferase (ALT). In addition, levels of key biochemical indicators, such as SOD, CAT, and total antioxidant capacity (T-AOC), were significantly enhanced, whereas hepatopancreas malondialdehyde levels and plasma pH, NH3, GST, and ALT levels were significantly decreased after rHMCs27 treatment followed by high ammonia stress. Moreover, high ammonia stress induced hepatopancreas tissue injury and apoptosis, but rHMCs27 treatment ameliorated these effects. Collectively, the current study revealed that in response to high ammonia stress, shrimp generate functional peptides, such as peptide HMCs27 from hemocyanin, which helps to attenuate the ammonia toxicity by enhancing the antioxidant system and the tricarboxylic acid cycle to decrease plasma NH3 levels and pH.

Endogenous enzymatic antioxidant status of whiteleg shrimp (Litopenaeus vannamei) following exposure to sublethal concentrations of silver nanoparticles.

Immunotoxicity of silver nanoparticles (AgNPs) to whiteleg shrimp (Litopenaeus vannamei) was assessed using redox-status orchestrating enzymes. To this end, the shrimp was exposed to sublethal AgNPs concentrations (0 % LC50: control; 25 % LC50: 0.97 mg/L; 50 % LC50: 1.95 mg/L; 75 % LC50: 2.92 mg/L). During the experiment, the behavior of superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), and glutathione peroxidase (GPx) was monitored, besides total antioxidant capacity (TAC) and malondialdehyde (MDA). The hepatopancreas SOD activity reduced about 63 %-76 % at.%50 LC50 and %75 LC50 AgNPs treatments, and CAT decreased in both tissues at 50 % LC50 AgNPs. TAC exhibited a U-form response in the hepatopancreas organ against stress caused by AgNPs, and hepatopancreas MDA displayed a time-dependent increase. Taken together, AgNPs triggered severe immunotoxicity through suppression of CAT, SOD, and TAC in the hepatopancreas tissue.

Thermal acclimation capacity and standard metabolism of the Pacific white shrimp Litopenaeus vannamei (Boone, 1931) at different temperature and salinity combinations.

In aquatic environments, rising temperatures reduce the oxygen content of the water while increasing the oxygen demand of organisms. In intensive shrimp culture, it is of great importance to know the thermal tolerance of cultured species and their oxygen consumption since this affects the physiological condition. In this study, the thermal tolerance of Litopenaeus vannamei was determined by dynamic and static thermal methodologies at different acclimation temperatures (15, 20, 25, and 30 °C) and salinities (10, 20, and 30 ppt). The oxygen consumption rate (OCR) was also measured to determine the standard metabolic rate (SMR) of shrimp. Acclimation temperature significantly affected the thermal tolerance and SMR of Litopenaeus vannamei (P < 0.01). Salinity had a large effect on SMR (P < 0.01) but did not influence the thermal acclimation of the shrimp (P > 0.01). Litopenaeus vannamei is a species that has high thermal tolerance and can survive at extreme temperatures (CTmin-CTmax: 7.2-41.9 °C) with its large dynamic (988, 992, and 1004 °C2) and static thermal polygon areas (748, 778 and 777 °C2) developed at the above temperature and salinity combinations and resistance zone (1001, 81 and 82 °C2). The optimal temperature range of Litopenaeus vannamei is the 25-30 °C range, where a decrease in standard metabolism is determined with increasing temperature. Given the SMR and optimal temperature range, the results of this study indicate that Litopenaeus vannamei should be cultured at 25-30 °C for effective production.

The effect of air exposure and re-water on gill microstructure and molecular regulation of Pacific white shrimp Penaeus vannamei.

The Penaeus vannamei is an important shrimp species with enormous commercial and ecological values. In production process, the air exposure resistance is vital for live transportation without water. We tested the air exposure resistant ability of P. vannamei, and carried out gill histological observation and gene expression analysis. The physiology and molecular response to the air exposure stress of P. vannamei was revealed. We found that body weight could affect the air exposure tolerance. Air exposure caused epithelial cell of gill filament shrinking and tissue fluid exudation within half of hour, and triggered oxidative stress response. After retrieved to water, epithelial cell shrinking and tissue fluid exudation recovered gradually, but oxidative and antioxidant response is still going on. Organisms reduced oxidative stress by regulating levels of antioxidants and antioxidant enzymes that remove reactive oxygen species (ROS) and RNA and DNA processing to repair tissue damage, and expression of apoptosis associated-genes altered. Furthermore, the survive shrimps could live steadily more than 5 days, and their gill filament recovered to normal state, proving that the damage of air exposure is reversible. These findings could be considered in the waterless live transportation of P. vannamei.

Role of myo-inositol supplementation against toxicity of excessive dietary copper in Pacific white shrimp Litopenaeus vannamei.

Raw materials for making dried shrimp (a type of foodstuff) are mostly from farmed shrimp and preliminary findings indicated that head copper (Cu) concentrations in some commercial dried shrimp products exceeded the safe limit specified in pollution-free aquatic products (50 mg/kg), which may influence food safety. Therefore, a 63-day feeding trial was conducted to explore effects of dietary Cu concentrations on accumulation of Cu in tissues, growth performance, immune response and antioxidant status of Pacific white shrimp (Litopenaeus vannamei). Moderating effect of myo-inositol (MI, adding 200 mg/kg diet) on the adverse impacts caused by excessive dietary Cu was also investigated. 600 shrimp (initial weight: 0.89 ± 0.00 g) were divided into five groups: 37.08 mg Cu/kg diet group (control group), 62.57 mg Cu/kg diet group, 125.99 mg Cu/kg diet group, 63.41 mg Cu/kg diet group (supplemented with MI) and 119.19 mg Cu/kg diet group (supplemented with MI). The results showed that dietary Cu concentrations increased from 37.08 to over 62.57 mg/kg, hepatopancreas Cu concentrations raised from 29.04 to 233.43-263.65 mg/kg, and muscle Cu concentrations only increased from 6.22 to 6.99-8.39 mg/kg. Report to control group, excessive Cu concentration (125.99 mg/kg) didn't significantly affect growth performance, but it notably reduced whole body lipid content and immune response, induced oxidative stress and damaged the hepatopancreas structure, which was ameliorated by MI supplementation. The results suggested that consuming shrimp head and its processed products weren't recommended. Cu concentrations of commercial feeds for Pacific white shrimp should be controlled below 62.57 mg/kg. Additionally, MI supplementation mitigated the negative impacts induced by excessive dietary Cu.

Silencing of Nrf2 in Litopenaeus vannamei, decreased the antioxidant capacity, and increased apoptosis and autophagy.

Nuclear factor E2-related factor 2 (Nrf2) is a multifunctional transcription factor that plays an important role in antioxidant activities. However, its effect on antioxidant capacity in Litopenaeus vannamei, an economically important crustacean, remains unclear. In this study, the role of Nrf2 in response to oxidative stress in L. vannamei was determined by its effect on relevant gene expression and enzymatic activity. Nrf2 was cloned and analyzed. Results revealed that Nrf2 contains a 1575 bp open reading frame encoding 524 amino acids and a conserved bZIP Maf domain. The sequence similarity of Nrf2 between L. vannamei and Homarus americanus is 81%. Although the Nrf2 expression was detected in all tissues, the Nrf2 expression levels were the highest in the hepatopancreas, followed by the eyestalk and muscle. RNA interference significantly decreased the expression of antioxidant-related genes (SOD, GPX, CAT, Trx, and HO-1; p < 0.05), significantly upregulated the expression of autophagy genes (Atg3, Atg4, Atg5, Atg10, and Atg12; p < 0.05) and apoptosis genes (Caspase-3 and P53; p < 0.05). Moreover, SOD, CAT, and GPX enzyme activities decreased whereas the MDA activity increased. The histological results of the shrimp injected with dsRNA-Nrf2 showed that the hepatic tubules were irregularly arranged, the lumen was abnormal, and a few hepatic tubules were significantly enlarged compared with those of the dsRNA-EGFP group. The hepatocytes were also vacuolated. In conclusion, this study provided evidence that Nrf2 is involved in the regulation of antioxidant capacity, oxidative stress, apoptosis, and autophagy in shrimp.

Energy metabolism response of Litopenaeus vannamei to combined stress of acute cold exposure and waterless duration: Implications for physiological regulation and waterless live transport.

Maintaining the homeostasis of energy metabolism is crucial for organism's stress tolerance and survival. Acute cold exposure (AC) and waterless duration (WD) represent the two predominate abiotic stressors during waterless live transport of Litopenaeus vannamei. Although previous reports have explored the physiological response of L. vannamei to combined stress AC + WD, the roles of energy metabolism response in regulation of stress tolerance remains unknown. The present study comparatively examined the variations of energy metabolism-related indicators in hemolymph (cortisol, hemocyanin, glucose and lactate), hepatopancreas and muscle tissues (levels of lactate and glycogen, activities of hexokinase (HK), phosphofructokinase (PFK), pyruvate kinase (PK), lactate dehydrogenase (LDH), succinate dehydrogenase (SDH) and adenosine triphosphatase (ATPase), and ATP levels). Combined stress significantly disturbed the homeostasis of energy metabolism with the increase in levels of hemocyanin, glucose and lactate, and decrease in glycogen and ATP content (P < 0.05). In addition, the activities of HK, PFK, PK, and SDH initially elevated and then decreased with the prolongation of combined stress from 3h to 9h duration, while the activity of lactate dehydrogenase (LDH) remained gradual elevation and ATPase activity decreased in a duration time dependent manner throughout the experiment. These alterations revealed that exposure to combined stress could accelerate anaerobic metabolism at initial stage and inhibit aerobic metabolism in a duration time-dependent manner, following with the reduction of energy biosynthesis and the disturbance of energy metabolism equilibrium. On the other hand, the progressive impairment on hepatopancreas tissue was observed under combined stress. In summary, the deficiency of ATP supply and histopathological injures on hepatopancreas tissue might the underlying mechanisms inducing mortality of L. vannamei during live transport.

Insights into the response mechanism of Litopenaeus vannamei exposed to cold stress during live transport combining untargeted metabolomics and biochemical assays.

Unfavorable conditions severely affect the survival quality of Litopenaeus vannamei (L. vannamei) during live transport and the molecular response mechanism needs to be clarified. In this study, metabolomics combined with conventional assay on physiological and histopathological responses were applied to deepen the understanding of L. vannamei to cold stress and provide. A solid foundation for regulation of transportation management. Physiological and biochemical analysis revealed the significant disturbance of glycolysis in hepatopancreas tissue. Furthermore, metabolomics based on the technique of ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry identified the significantly differential metabolites between acute cold exposure (AC) and normal control (NC) groups. Moreover, KEEG result indicated that the pathways of amino acid, glycerophospholipid, and nuclei acid metabolism and ABC transporters in hepatopancreas were significantly disturbed. Furthermore, histopathology and ultrastructure verified the impairment in hepatopancreas during cold stress. Overall, the concurrent use of metabolomics and biochemical assays was demonstrated to be sensitive and effective in providing new insights into the response mechanism of L. vannamei to cold stress.